Soybean resistant to cyst nematodes

ABSTRACT

A transgenic soybean plant or parts thereof, resistant to soybean cyst nematodes, transformed to express Glyma18g02570, Glyma18g02580, or Glyma18g02590, or a variant thereof. Also provided is a method of making such a plant. Also provided is an artificial DNA construct encoding Glyma18g02570, Glyma18g02580, or Glyma18g02590, or a variant thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional Application No. 61/799,912 filed 15 Mar. 2013, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant number 0820642 awarded by National Science Foundation Plant Genome Research Program and DBI-0845196 awarded by National Science Foundation. The government has certain rights in the invention.

MATERIAL INCORPORATED-BY-REFERENCE

The Sequence Listing, which is a part of the present disclosure, includes a computer readable form comprising nucleotide and/or amino acid sequences of the present invention. The subject matter of the Sequence Listing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Soybean (Glycine max (L.) Merr.) is a major crop that provides a sustainable source of protein and oil worldwide. Soybean cyst nematode (SCN), Heterodera glycines Ichinohe, is a major constraint to soybean production. This nematode causes more than $1 billion in yield losses annually in the United States alone, making it the most economically important pathogen of soybeans. Although planting of resistant cultivars forms the core management strategy for this pathogen, nothing is known about the nature of resistance. Moreover, the increase in virulent populations of this parasite on most known resistance sources necessitates the development of novel approaches for control.

SUMMARY OF THE INVENTION

Disclosed herein are methods of transforming a soybean plant using artificial DNA constructs to increase resistance to soybean cyst nematode (SCN).

One aspect provides a transgenic soybean resistant to SCN, or a seed, plant part, or progeny thereof. In some embodiments, the soybean plant can be transformed with an artificial DNA construct. In some embodiments, the DNA construct includes, as operably associated components in the 5′ to 3′ direction of transcription, a promoter that functions in a soybean. In some embodiments, the DNA construct also includes a transcribable nucleic acid molecule.

In some embodiments, the transcribable nucleic acid molecule includes a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 (Glyma18g02570), SEQ ID NO: 2 (Glyma18g02580), and SEQ ID NO: 3 (Glyma18g02590). In some embodiments, the transcribable nucleic acid molecule includes a nucleotide sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 encoding a polypeptide having Glyma18g02570, Glyma18g02580, or Glyma18g02590 activity, respectively.

In some embodiments, the transcribable nucleic acid molecule includes a nucleotide sequence encoding a polypeptide comprising SEQ ID NO: 4 (Glyma18g02570), SEQ ID NO: 5 (Glyma18g02580), SEQ ID NO: 6 (Glyma18g02590), and SEQ ID NO: 7 (Forrest SNAP A111D mutant). In some embodiments, the transcribable nucleic acid molecule includes a nucleotide sequence encoding a polypeptide having an amino acid sequence at least 95% identical a polypeptide comprising SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 having Glyma18g02570, Glyma18g02580, Glyma18g02590, or SNAP activity, respectively.

In some embodiments, the transcribable nucleic acid molecule includes a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the polynucleotide encodes a polypeptide having Glyma18g02570, Glyma18g02580, or Glyma18g02590 activity. In some embodiments, stringent conditions include incubation at 65° C. in a solution including 6×SSC (0.9 M sodium chloride and 0.09 M sodium citrate). In some embodiments, the transcribable nucleic acid molecule includes a nucleotide sequence which is the reverse complement of nucleotide sequences disclosed herein.

In some embodiments, the DNA construct also includes a transcriptional termination sequence. In some embodiments, the transgenic soybean exhibits increased SCN resistance compared to a control not expressing the transcribable nucleic acid molecule.

In some embodiments, the nucleotide sequence can be at least 95% identical to SEQ ID NO: 3 having one of more mutations selected from the group consisting of C163225G, G174968T, A164972AGGT, C164974A, C163208A, G164965C, G164968C, A164972AGGC, and C164974A. In some embodiments, the encoded polypeptide includes an amino acid sequence at least 95% identical to SEQ ID NO: 6 having one of more mutations selected from the group consisting of D208E, D286Y, D287E, -288V, L289I, Q203K, E285Q, D286H, D287E, -288A, L289I, and A111D.

In some embodiments, the encoded polypeptide includes an amino acid sequence at least 95% identical to SEQ ID NO: 6, a mutation of A111D, and Glyma18g02590 polypeptide activity. In some embodiments, the transcribable nucleic acid molecule is expressed in epidermis, vascular tissue, meristem, cambium, cortex, pith, leaf, sheath, root, flower, developing ovule or seed.

In some embodiments, the promoter includes an inducible promoter or a tissue-specific promoter. In some embodiments, the promoter includes a nematode-inducible promoter. In some embodiments, the promoter is selected from the group consisting of factor EF1α gene promoter; rice tungro bacilliform virus (RTBV) gene promoter; cestrum yellow leaf curling virus (CmYLCV) promoter; tCUP cryptic promoter system; T6P-3 promoter; S-adenosyl-L-methionine synthetase promoter; Raspberry E4 gene promoter; cauliflower mosaic virus 35S promoter; figwort mosaic virus promoter; conditional heat-shock promoter; promoter sub-fragments of sugar beet V-type H+-ATPase subunit c isoform; and beta-tubulin promoter.

In some embodiments, increased SCN resistance comprises at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1000% decrease in susceptibility to SCN as compared to a non-transformed control.

In some embodiments, the transcribable nucleic acid molecule includes a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In some embodiments, the transcribable nucleic acid molecule includes a nucleotide sequence at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and encodes a polypeptide having Glyma18g02570, Glyma18g02580, or Glyma18g02590 activity, respectively. In some embodiments, the transcribable nucleic acid molecule encodes a polypeptide including SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. In some embodiments, the transcribable nucleic acid molecule encodes a polypeptide including an amino acid sequence at least 95% identical to SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 and having Glyma18g02570, Glyma18g02580, Glyma18g02590, or SNAP activity, respectively.

In some embodiments, the transgenic progeny, seed, or part comprises the transcribable nucleic acid molecule.

One aspect provides a soybean plant including in its genome at least one introgressed allele locus associated with an SCN resistant phenotype. In some embodiments, the locus can be in a genomic region flanked by at least two loci selected from TABLE 6. In some embodiments, the soybean plant also includes in its genome one or more polymorphic loci including alleles or combinations of alleles that are not found in an SCN resistant variety and that are linked to said locus associated with an SCN resistant phenotype, or a progeny plant therefrom. In some embodiments, the at least one allele locus is selected from the group consisting of Glyma18g02570, Glyma18g02580, and Glyma18g02590.

One aspect provides a method of producing a soybean plant as disclosed herein including crossing a first soybean plant lacking a locus associated with an SCN resistant phenotype with a second soybean plant. In some embodiments, the second soybean plant includes an allele of at least one polymorphic nucleic acid associated with an SCN resistant phenotype located in a genomic region flanked by at least two loci selected from TABLE 6. In some embodiments, the second soybean plant also includes at least one additional polymorphic locus located outside of said region that is not present in the first soybean plant, to obtain a population of soybean plants segregating for the polymorphic locus associated with an SCN resistant phenotype and said additional polymorphic locus.

In some embodiments, the method also includes detecting said polymorphic locus in at least one soybean plant from said population of soybean plants. In some embodiments, the method also includes selecting a soybean plant including the locus associated with an SCN resistant phenotype that lacks the additional polymorphic locus, thereby obtaining a soybean plant including in its genome at least one introgressed allele of a polymorphic nucleic acid associated with an SCN resistant phenotype. In some embodiments, the first soybean plant includes germplasm capable of conferring agronomically elite characteristics to a progeny plant of the first soybean plant and the second soybean plant.

One aspect provides an artificial DNA construct including, as operably associated components in the 5′ to 3′ direction of transcription, a promoter that functions in a soybean.

In some embodiments, the DNA construct also includes a transcribable nucleic acid molecule. In some embodiments, the transcribable nucleic acid molecule includes a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, or a nucleotide sequence at least 95% identical thereto encoding a polypeptide having Glyma18g02570, Glyma18g02580, or Glyma18g02590 activity, respectively. In some embodiments, the transcribable nucleic acid molecule includes a nucleotide sequence encoding a polypeptide including SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or an amino acid sequence at least 95% identical thereto having Glyma18g02570, Glyma18g02580, Glyma18g02590, or SNAP activity, respectively. In some embodiments, the transcribable nucleic acid molecule includes a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the polynucleotide encodes a polypeptide having Glyma18g02570, Glyma18g02580, or Glyma18g02590 activity. In some embodiments, said stringent conditions include incubation at 65° C. in a solution including 6×SSC (0.9 M sodium chloride and 0.09 M sodium citrate). In some embodiments, the transcribable nucleic acid molecule includes a nucleotide sequence which is the reverse complement of nucleotide sequences disclosed herein.

In some embodiments, DNA construct also includes a transcriptional termination sequence.

One aspect provides a method of increasing SCN resistance of a soybean including transforming a soybean plant with an artificial DNA construct disclosed herein.

DESCRIPTION OF THE DRAWINGS

Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

FIG. 1 is a series of drawings and a sequence listing illustrating the positional cloning of the Rhg1 gene. FIG. 1A shows high-density genetic maps of the Rhg1 locus developed using two recombinant inbred line populations, developed from crosses between a resistant line “Forrest” (F) and a susceptible line “Essex” (E) or “Williams 82” (W), showing recombinant lines WxF6034 (I, SCN-susceptible), ExF3126 (II, SCN-resistant) and ExF4361 (III, SCN-resistant). Black horizontal lines represent approximately 370 kb of the Rhg1 chromosomal interval. Arrows designate DNA marker positions and names. Numbers above the black horizontal line denote marker position relative to marker RLK (an LRR-RLK gene at the Rhg1 locus; assigned position ‘0’). Arrows with one asterisk designate the physical position of each tested DNA marker within the Rhg1 locus using published DNA sequence of Williams 82 as a reference. Arrows with no asterisk represent the DNA markers with Forrest alleles found in recombinants WxF6034, ExF3126 and ExF4361. Arrows with two asterisks represent the DNA markers with heterozygote alleles (Forrest allele with Essex allele). The arrow with three asterisks represents DNA marker 600 having polymorphisms between Essex and Forrest, and between Essex and Williams 82, but not between Forrest and Williams 82. FIG. 1B shows the genomic DNA gene model for the SNAP gene. The gene is 4,223 bp from start codon to stop codon and contains nine exons (light-grey boxes) and eight introns (solid black lines). Numbers above the light-grey boxes and solid black line indicate the length (bp) of each exon or intron, while the numbers under the dotted lines indicate the nucleotide position relative to the first nucleotide of the start codon. FIG. 1C shows a comparison of the predicted SNAP protein sequences between Forrest and Essex with the amino acid differences (Y206D, E207D, V288- and I289L) highlighted. FIG. 1D shows the predicted armadillo/beta-catenin-like repeat sequence (marker 570). FIG. 1E shows the predicted amino acid transporter sequence (marker 580).

FIG. 2 is a schematic representation of the amino acid differences in the predicted SNAP protein sequences in 11 soybean lines with the number indicating amino acid position. Amino acid differences detected in the exons between Forrest and Essex are boxed along with the special amino acids of the PI88788-type PIs in SNAP. Amino acids marked in the boxes are the different amino acids between Peking type SNAP, PI88788 type SNAP, and susceptible type SNAP.

FIG. 3 shows images of a soybean plant. FIG. 3A and FIG. 3B show the virus-induced gene-silencing (VIGS) phenotype of Glyma18g02590-VIGS-AS bombarded plants at 16 days post-inoculation. FIG. 3C and FIG. 3D show the VIGS phenotype of Glyma18g02590-VIGS-AS rub-inoculated plants at 16 days post-inoculation.

FIG. 4 is Venn diagram illustrating the overlapping SNPs, insertions, and deletions conferring resistance of Rhg1 to SCN. The number following S, I, and D represents the number of SNPs (S), insertions (I), or deletions (D). EFP88, EFP, EF88, EP88, FP88, EP, E88, FP, F88, and P88 represent the overlapping SNPs, insertions, or deletions of Essex (E), Forrest (F), Peking (P), and PI88788 (88); Essex, Forrest, and PI88788; Essex, Forrest, and PI88788; Essex, Peking, and P188788; Forrest, Peking, and P188788; Essex and Peking; Essex and P188788; Forrest and Peking; Forrest and PI88788; and Peking and PI88788, respectively.

FIG. 5 is a histogram illustrating SCN susceptibility of Forrest SNAP Type III mutant A111D, as compared to SCN-resistant wild-type Forrest.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is based, at least in part, on the discovery that three genes mapped to the Rhg1 (for resistance to Heterodera glycines 1) locus confer resistance to SCN.

Reported herein is the map-based cloning of three genes at the Rhg1 locus, a major quantitative trait locus conferring resistance to this pathogen. Results herein indicate that three genes that can confer SCN-resistance at the Rhg1 locus include Glyma18g02570 (an armadillo/beta-catenin-like repeat), Glyma18g02580 (an amino acid transporter), or Glyma18g02590 (a SNAP-like protein).

According to the approach described herein, a soybean cell or plant can be transformed so as to provide for SCN resistance. In some embodiments, a soybean host cell or plant can be transformed with a nucleic acid molecule encoding a polypeptide having activity of Glyma18g02570, Glyma18g02580, or Glyma18g02590. A nucleic acid encoding a polypeptide having Glyma18g02570, Glyma18g02580, or Glyma18g02590 activity can confer SCN resistance.

Since the discovery of the genes involved in resistance to SCN, others have published data providing confirmation that the three genes are involved in the resistance to SCN. Proof of principle data includes the following additional evidence that the Rhg1 locus confers SCN-resistance in soybean. It has since been shown that upregulation of genes at nematode feeding sites in near-isogenic lines of resistant and susceptible soybean cultivars differ at the Rhg1 locus (Kandoth et al., Plant Physiology, 155:1960-1975, 2011). These results show that expression of Glyma18g02580 and Glyma18g02590 increased in resistant cells as described herein (see e.g., TABLE 1). The effect of copy number variation of multiple genes at the Rhg1 locus was shown for nematode resistance in soybean (Cook et al., Science, 338(6111):1206-1209, 2012).

TABLE 1 Glyma18g02580 and Glyma18g02590 gene expression in SCN-resistant cells. Gene Description Fold increase (R:S) Glyma18g02580.1 Amino acid transporter 4.08 Glyma18g02590.1 NSF soluble attachment 1.50 protein

TRANSFORMED ORGANISM

Provided herein is a soybean plant genetically engineered to be SCN-resistant. The host genetically engineered to resist SCN can be any soybean plant or cell.

Assays to assess SCN resistance are well known in the art (see e.g., Examples). Therefore, except as otherwise noted herein, plant SCN resistance can be carried out in accordance with such assays.

One aspect of the current invention is therefore directed to the aforementioned plants, and parts thereof, and methods for using these plants and plant parts. Plant parts include, but are not limited to, pollen, an ovule, and a cell. The invention further provides tissue cultures of regenerable cells of these plants, which cultures regenerate soybean plants capable of expressing all the physiological and morphological characteristics of the starting variety. Such regenerable cells may include embryos, meristematic cells, pollen, leaves, roots, root tips or flowers, or protoplasts or callus derived therefrom. Also provided by the invention are soybean plants regenerated from such a tissue culture, wherein the plants are capable of expressing all the physiological and morphological characteristics of the starting plant variety from which the regenerable cells were obtained.

Such SCN-resistant plants can have a commercially significant yield, for example, a yield of at least 90% to at least 110% (e.g., at least 95%, 100%, 105%) of a soybean check line. Plants are provided comprising the Glyma18g02570, Glyma18g02580, or Glyma18g02590 alleles and SCN resistance and a grain yield of at least about 90%, 94%, 98%, 100%, 105% or about 110% of these lines.

In various embodiments, a nucleic acid sequence encoding a polypeptide having Glyma18g02570, Glyma18g02580, or Glyma18g02590 activity is engineered in a host plant (e.g., a soybean plant) so as to result in an SCN-resistant phenotype. A nucleic acid sequence encoding a polypeptide having Glyma18g02570, Glyma18g02580, or Glyma18g02590 activity can be endogenous or exogenous to the host plant. Transformation of a plant to express a polypeptide having Glyma18g02570, Glyma18g02580, or Glyma18g02590 activity can convey SCN resistance to a host lacking such phenotype. Transformation of a plant to express a polypeptide having Glyma18g02570, Glyma18g02580, or Glyma18g02590 activity can increase SCN resistance to a host already possessing such phenotype.

In some embodiments, a host plant transformed to express a polypeptide having Glyma18g02570, Glyma18g02580, or Glyma18g02590 activity can exhibit at least about 10% decrease in susceptibility to SCN. For example, a host plant transformed to express a polypeptide having Glyma18g02570, Glyma18g02580, or Glyma18g02590 activity can exhibit at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% decrease in susceptibility to SCN as compared to a non-transformed control. As another example, a host plant transformed to express a polypeptide having Glyma18g02570, Glyma18g02580, or Glyma18g02590 activity can exhibit at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1000% decrease in susceptibility to SCN as compared to a non-transformed control.

Genes of particular interest for engineering a soybean plant to exhibit SCN resistance include Glyma18g02570 (SEQ ID NO: 1), Glyma18g02580 (SEQ ID NO: 2), or Glyma18g02590 (SEQ ID NO: 3). As described herein, Glyma18g02570, Glyma18g02580, or Glyma18g02590 have been mapped to the Rhg1 locus and can confer SCN-resistance.

A transformed host soybean plant can comprise a nucleotide sequence of SEQ ID NO: 1 (Glyma18g02570), SEQ ID NO: 2 (Glyma18g02580), or SEQ ID NO: 3 (Glyma18g02590). A transformed host soybean plant can comprise a nucleotide sequence having at least about 80% sequence identity to SEQ ID NO: 1 (Glyma18g02570), SEQ ID NO: 2 (Glyma18g02580), or SEQ ID NO: 3 (Glyma18g02590), wherein the nucleotide sequence encodes a polypeptide having Glyma18g02570, Glyma18g02580, or Glyma18g02590 activity, respectively, or the transformed soybean exhibits SCN resistance. For example, a transformed host soybean plant can comprise a nucleotide sequence having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity to SEQ ID NO: 1 (Glyma18g02570), SEQ ID NO: 2 (Glyma18g02580), or SEQ ID NO: 3 (Glyma18g02590), wherein the nucleotide sequence encodes a polypeptide having Glyma18g02570, Glyma18g02580, or Glyma18g02590 activity, respectively, or the transformed soybean exhibits SCN resistance.

A nucleotide sequence described herein can include one or mutations affecting the activity of a Glyma18g02570, Glyma18g02580, or Glyma18g02590 polypeptide or host SCN resistance. For example, a nucleotide sequence of SEQ ID NO: 1 (Glyma18g02570), SEQ ID NO: 2 (Glyma18g02580), or SEQ ID NO: 3 (Glyma18g02590) can have one or more mutations affecting the activity of a Glyma18g02570, Glyma18g02580, or Glyma18g02590 polypeptide or host SCN resistance. For example, a nucleotide sequence variant (e.g., at least 80%, 85%, 90%, 95, or 99% identity) of SEQ ID NO: 3 (Glyma18g02590) can have one or more of the following mutations: C163225G, G174968T, A164972AGGT, C164974A, C163208A, G164965C, G164968C, A164972AGGC, or C164974A. As another example, the SNAP gene (Glyma18g02590; e.g., SEQ ID NO: 3) in Forrest or Peking backgrounds can include one or more of the following mutations: C163225G, G174968T, A164972AGGT, or C164974A. As another example, the SNAP gene (Glyma18g02590; e.g., SEQ ID NO: 3) in a PI88788 background can include one or more of the following mutations: C163208A, G164965C, G164968C, A164972AGGC, or C164974A.

A transformed host soybean plant can comprise a nucleotide sequence encoding a polypeptide of SEQ ID NO: 4 (Glyma18g02570), SEQ ID NO: 5 (Glyma18g02580), SEQ ID NO: 6 (Glyma18g02590), or SEQ ID NO: 7 (Forrest SNAP A111D mutant). A transformed host soybean plant can comprise a nucleotide sequence encoding a polypeptide having at least about 80% sequence identity to SEQ ID NO: 4 (Glyma18g02570), SEQ ID NO: 5 (Glyma18g02580), SEQ ID NO: 6 (Glyma18g02590), or SEQ ID NO: 7 (Forrest SNAP A111D mutant), wherein the polypeptide has Glyma18g02570, Glyma18g02580, Glyma18g02590, or SNAP activity, respectively, or the transformed soybean exhibits SCN resistance. For example, a transformed host soybean plant can comprise a nucleotide sequence encoding a polypeptide having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity to SEQ ID NO: 4 (Glyma18g02570), SEQ ID NO: 5 (Glyma18g02580), SEQ ID NO: 6 (Glyma18g02590), or SEQ ID NO: 7 (Forrest SNAP A111D mutant), wherein the nucleotide sequence encodes a polypeptide having Glyma18g02570, Glyma18g02580, Glyma18g02590, or SNAP activity, respectively, or the transformed soybean exhibits SCN resistance.

A polypeptide sequence described herein can include one or mutations affecting the activity of a Glyma18g02570, Glyma18g02580, or Glyma18g02590 polypeptide or host SCN resistance. For example, an encoded or expressed polypeptide of SEQ ID NO: 4 (Glyma18g02570), SEQ ID NO: 5 (Glyma18g02580), SEQ ID NO: 6 (Glyma18g02590), or SEQ ID NO: 7 (Forrest SNAP A111D mutant) can have one or more mutations affecting the activity of the polypeptide or host SCN resistance. For example, an encoded or expressed polypeptide variant (e.g., at least 80%, 85%, 90%, 95, or 99% identity) of SEQ ID NO: 3 (Glyma18g02590) can have one or more of the following mutations: D208E, D286Y, D287E, -288V, L289I, Q203K, E285Q, D286H, D287E, -288A, L289I, or A111D. As another example, the SNAP-like protein (SEQ ID NO: 6, encoded by, e.g., Glyma18g02590, SEQ ID NO: 3) in Forrest or Peking backgrounds can include one or more of the following mutations: D208E, D286Y, D287E, -288V, L289I, or A111D. As another example, the SNAP-like protein (SEQ ID NO: 6, encoded by, e.g., Glyma18g02590, SEQ ID NO: 3) in a PI88788 background can include one or more of the following mutations: Q203K, E285Q, D286H, D287E, -288A, L289I, or A111D.

As another example, a transformed soybean can comprise a nucleotide sequence that hybridizes under stringent conditions to a Glyma18g02570, Glyma18g02580, or Glyma18g02590 polynucleotide (e.g., SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, respectively) over the entire length thereof, and which encodes a polypeptide having Glyma18g02570, Glyma18g02580, Glyma18g02590, or SNAP A111D mutant (e.g., SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, respectively) activity.

As a further example, a transformed soybean can comprise the complement to any of the above sequences.

Variant Sequences

As describe above, a plant can be transformed with a variant of a Glyma18g02570, Glyma18g02580, or Glyma18g02590 polynucleotide (e.g., SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3) or with a polynucleotide encoding a variant of a Glyma18g02570, Glyma18g02580, Glyma18g02590, SNAP A111D mutant (e.g., SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, respectively) polypeptide. These species SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and their corresponding encoded polypeptides, are representative of the genus of variant nucleic acid and polypeptides, respectively, because all variants must possess the specified catalytic activity (e.g., Glyma18g02570, Glyma18g02580, or Glyma18g02590 activity) and must have the percent identity required above to the reference sequence.

Promoters

One or more of the nucleotide sequences discussed above (e.g., Glyma18g02570, Glyma18g02580, or Glyma18g02590 or a variant thereof) can be operably linked to a promoter that can function in a plant, such as soybean. Promoter selection can allow expression of a desired gene product under a variety of conditions.

Promoters can be selected for optimal function in a soybean host cell into which the vector construct will be inserted. Promoters can also be selected on the basis of their regulatory features. Examples of such features include enhancement of transcriptional activity and inducibility.

Numerous promoters functional in a soybean plant will be known to one of skill in the art (see, e.g., Weise et al., Applied Microbiology and Biotechnology, 70(3):337-345, 2006; Saidi et al., Plant Molecular Biology, 59(5):697-711, 2005; Horstmann et al., BMC Biotechnology, 4, 2004; Holtorf et al., Plant Cell Reports, 21(4):341-346, 2002; Zeidler et al., Plant Molecular Biology, 30(1):199-205, 1996). Except as otherwise noted herein, therefore, the processes and compositions of the present disclosure can be carried out in accordance with such known promoters. Examples of promoters than can be used in accord with methods and compositions described herein include, but are not limited to, factor EF1α gene promoter (US App Pub No. 2008/0313776); rice tungro bacilliform virus (RTBV) gene promoter (US App Pub No. 2008/0282431); cestrum yellow leaf curling virus (CmYLCV) promoter (Stavolone et al., Plant Molecular Biology, 53(5):663-673, 2003); tCUP cryptic promoter system (Malik et al., Theoretical and Applied Genetics, 105(4):505-514, 2002); T6P-3 promoter (JP2002238564); S-adenosyl-L-methionine synthetase promoter (WO/2000/037662); Raspberry E4 gene promoter (U.S. Pat. No. 6,054,635); cauliflower mosaic virus 35S promoter (Benfey et al., Science, 250(4983):959-966, 1990); figwort mosaic virus promoter (U.S. Pat. No. 5,378,619); conditional heat-shock promoter (Saidi et al., Plant Molecular Biology, 59(5):697-711, 2005); promoter sub-fragments of the sugar beet V-type H+-ATPase subunit c isoform (Holtorf et al., Plant Cell Reports, 21(4):341-346, 2002); beta-tubulin promoter (Jost et al., Current Genetics, 47(2):111-120, 2005); and bacterial quorum-sensing components (You et al., Plant Physiology, 140(4):1205-1212, 2006).

The promoter can be an inducible promoter. For example, the promoter can be induced according to temperature, pH, a hormone, a metabolite (e.g., lactose, mannitol, an amino acid), light (e.g., wavelength specific), osmotic potential (e.g., salt induced), a heavy metal, or an antibiotic. As another example, the promoter can be a nematode-inducible promoter, such as pZF (Kandoth et al. Plant Physiol. 155:1960-1975 (2011)). Numerous standard inducible promoters will be known to one of skill in the art.

The term “chimeric” is understood to refer to the product of the fusion of portions of two or more different polynucleotide molecules. “Chimeric promoter” is understood to refer to a promoter produced through the manipulation of known promoters or other polynucleotide molecules. Such chimeric promoters can combine enhancer domains that can confer or modulate gene expression from one or more promoters or regulatory elements, for example, by fusing a heterologous enhancer domain from a first promoter to a second promoter with its own partial or complete regulatory elements. Thus, the design, construction, and use of chimeric promoters according to the methods disclosed herein for modulating the expression of operably linked polynucleotide sequences are encompassed by the present invention.

Novel chimeric promoters can be designed or engineered by a number of methods. For example, a chimeric promoter may be produced by fusing an enhancer domain from a first promoter to a second promoter. The resultant chimeric promoter may have novel expression properties relative to the first or second promoters. Novel chimeric promoters can be constructed such that the enhancer domain from a first promoter is fused at the 5′ end, at the 3′ end, or at any position internal to the second promoter.

Constructs

Any of the transcribable polynucleotide molecule sequences described above can be provided in a construct. Constructs of the present invention generally include a promoter functional in the host plant, such as soybean, operably linked to a transcribable polynucleotide molecule encoding a polypeptide with Glyma18g02570, Glyma18g02580, or Glyma18g02590 activity, such as provided in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or variants thereof as discussed above.

Exemplary promoters are discussed above. One or more additional promoters may also be provided in the recombinant construct. These promoters can be operably linked to any of the transcribable polynucleotide molecule sequences described above.

The term “construct” is understood to refer to any recombinant polynucleotide molecule such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a polynucleotide molecule where one or more polynucleotide molecule has been linked in a functionally operative manner, i.e. operably linked. The term “vector” or “vector construct” is understood to refer to any recombinant polynucleotide construct that may be used for the purpose of transformation, i.e., the introduction of heterologous DNA into a host plant, such as a soybean.

In addition, constructs may include, but are not limited to, additional polynucleotide molecules from an untranslated region of the gene of interest. These additional polynucleotide molecules can be derived from a source that is native or heterologous with respect to the other elements present in the construct.

Host cells developed according to the approaches described herein can be evaluated by a number of means known in the art (see, e.g., Studier, Protein Expr Purif, 41(1):207-234, 2005; Gellissen, ed., (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10:3527310363; Baneyx, (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10:0954523253).

Molecular Engineering

The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Green and Sambrook 2012 Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, ISBN-10: 1605500569; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

The terms “heterologous DNA sequence”, “exogenous DNA segment” or “heterologous nucleic acid,” as used herein, each refer to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides. A “homologous” DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.

Expression vector, expression construct, plasmid, or recombinant DNA construct is generally understood to refer to a nucleic acid that has been generated via human intervention, including by recombinant means or direct chemical synthesis, with a series of specified nucleic acid elements that permit transcription or translation of a particular nucleic acid in, for example, a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector can include a nucleic acid to be transcribed operably linked to a promoter.

A “promoter” is generally understood as a nucleic acid control sequence that directs transcription of a nucleic acid. An inducible promoter is generally understood as a promoter that mediates transcription of an operably linked gene in response to a particular stimulus. A promoter can include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter can optionally include distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

A “transcribable nucleic acid molecule” as used herein refers to any nucleic acid molecule capable of being transcribed into a RNA molecule. Methods are known for introducing constructs into a cell in such a manner that the transcribable nucleic acid molecule is transcribed into a functional mRNA molecule that is translated and therefore expressed as a protein product. Constructs may also be constructed to be capable of expressing antisense RNA molecules, in order to inhibit translation of a specific RNA molecule of interest. For the practice of the present disclosure, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art (see, e.g., Sambrook and Russell, (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10:0879697717; Ausubel et al., (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10:0471250929; Sambrook and Russell, (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10:0879695773; Elhai, J. and Wolk, C. P., Methods in Enzymology, 167:747-754, 1988).

The “transcription start site” or “initiation site” is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. With respect to this site all other sequences of the gene and its controlling regions can be numbered. Downstream sequences (i.e., further protein encoding sequences in the 3′ direction) can be denominated positive, while upstream sequences (mostly of the controlling regions in the 5′ direction) are denominated negative.

“Operably-linked” or “functionally linked” refers preferably to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be “operably linked to” or “associated with” a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation. The two nucleic acid molecules may be part of a single contiguous nucleic acid molecule and may be adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.

A “construct” is generally understood as any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating nucleic acid molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecule has been operably linked.

A constructs of the present disclosure can contain a promoter operably linked to a transcribable nucleic acid molecule operably linked to a 3′ transcription termination nucleic acid molecule. In addition, constructs can include but are not limited to additional regulatory nucleic acid molecules from, e.g., the 3′-untranslated region (3′ UTR). Constructs can include but are not limited to the 5′ untranslated regions (5′ UTR) of an mRNA nucleic acid molecule which can play an important role in translation initiation and can also be a genetic component in an expression construct. These additional upstream and downstream regulatory nucleic acid molecules may be derived from a source that is native or heterologous with respect to the other elements present on the promoter construct.

The term “transformation” refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as “transgenic” cells, and organisms comprising transgenic cells are referred to as “transgenic organisms”.

“Transformed,” “transgenic,” and “recombinant” refer to a host cell or organism such as a bacterium, cyanobacterium, animal or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome as generally known in the art. Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like. The term “untransformed” refers to normal cells that have not been through the transformation process.

“Wild-type” refers to a virus or organism found in nature without any known mutation.

Design, generation, and testing of the variant nucleotides, and their encoded polypeptides, having the above required percent identities and retaining a required activity of the expressed protein is within the skill of the art. For example, directed evolution and rapid isolation of mutants can be according to methods described in references including, but not limited to, Link et al., Nature Reviews, 5(9):680-688, 2007; Sanger et al., Gene, 97(1):119-123, 1991; and Ghadessy et al., Proc Natl Acad Sci USA, 98(8):4552-4557, 2001. Thus, one skilled in the art could generate a large number of nucleotide and/or polypeptide variants having, for example, at least 95-99% identity to the reference sequence described herein and screen such for desired phenotypes according to methods routine in the art.

Nucleotide and/or amino acid sequence identity percent (%) is understood as the percentage of nucleotide or amino acid residues that are identical with nucleotide or amino acid residues in a candidate sequence in comparison to a reference sequence when the two sequences are aligned. To determine percent identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum percent sequence identity. Sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software is used to align sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. When sequences are aligned, the percent sequence identity of a given sequence A to, with, or against a given sequence B (which can alternatively be phrased as a given sequence A that has or comprises a certain percent sequence identity to, with, or against a given sequence B) can be calculated as: percent sequence identity=X/Y100, where X is the number of residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

Generally, conservative substitutions can be made at any position so long as the required activity is retained. So-called conservative exchanges can be carried out in which the amino acid which is replaced has a similar property as the original amino acid, for example the exchange of Glu by Asp, Gln by Asn, Val by Ile, Leu by Ile, and Ser by Thr. Deletion is the replacement of an amino acid by a direct bond. Positions for deletions include the termini of a polypeptide and linkages between individual protein domains. Insertions are introductions of amino acids into the polypeptide chain, a direct bond formally being replaced by one or more amino acids. Amino acid sequence can be modulated with the help of art-known computer simulation programs that can produce a polypeptide with, for example, improved activity or altered regulation. On the basis of this artificially generated polypeptide sequences, a corresponding nucleic acid molecule coding for such a modulated polypeptide can be synthesized in-vitro using the specific codon-usage of the desired host cell.

“Highly stringent hybridization conditions” are defined as hybridization at 65° C. in a 6×SSC buffer (i.e., 0.9 M sodium chloride and 0.09 M sodium citrate). Given these conditions, a determination can be made as to whether a given set of sequences will hybridize by calculating the melting temperature (T_(m)) of a DNA duplex between the two sequences. If a particular duplex has a melting temperature lower than 65° C. in the salt conditions of a 6×SSC, then the two sequences will not hybridize. On the other hand, if the melting temperature is above 65° C. in the same salt conditions, then the sequences will hybridize. In general, the melting temperature for any hybridized DNA:DNA sequence can be determined using the following formula: T_(m)=81.5° C.+16.6(log₁₀[Na⁺])+0.41(fraction G/C content)−0.63(% formamide)−(600/1). Furthermore, the T_(m) of a DNA:DNA hybrid is decreased by 1-1.5° C. for every 1% decrease in nucleotide identity (see, e.g., Sambrook and Russell, (2006)).

Host cells can be transformed using a variety of standard techniques known to the art (see, e.g., Sambrook and Russell (2006); Ausubel et al. (2002); Sambrook and Russell, (2001); Elhai, J. and Wolk, C. P., 1988). Such techniques include, but are not limited to, viral infection, calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, receptor-mediated uptake, cell fusion, electroporation, and the like. The transfected cells can be selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome.

Exemplary nucleic acids which may be introduced to a host cell include, for example, DNA sequences or genes from another species, or even genes or sequences which originate with or are present in the same species, but are incorporated into recipient cells by genetic engineering methods. The term “exogenous” is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in the transforming DNA segment or gene, or genes which are normally present and that one desires to express in a manner that differs from the natural expression pattern, e.g., to over-express. Thus, the term “exogenous” gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell. The type of DNA included in the exogenous DNA can include DNA which is already present in the cell, DNA from another individual of the same type of organism, DNA from a different organism, or a DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.

Host strains developed according to the approaches described herein can be evaluated by a number of means known in the art (see, e.g., Studier, 2005; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

Methods of down-regulation or silencing genes are known in the art. For example, expressed protein activity can be down-regulated or eliminated using antisense oligonucleotides, protein aptamers, nucleotide aptamers, and RNA interference (RNAi) (e.g., small interfering RNAs (sRNA), short hairpin RNA (shRNA), and micro RNAs (miRNA) (see, e.g., Fanning and Symonds, Handb Exp Pharmacol., 173:289-303G, 2006, describing hammerhead ribozymes and small hairpin RNA; Helene, C., et al., Ann. N.Y. Acad. Sci., 660:27-36, 1992; Maher, Bioassays 14(12):807-15, 1992, describing targeting deoxyribonucleotide sequences; Lee et al., Curr Opin Chem Biol., 10:1-8, 2006, describing aptamers; Reynolds et al., Nature Biotechnology, 22(3):326-330, 2004, describing RNAi; Pushparaj and Melendez, Clin. and Exp. Pharm. and Phys., 33(5-6):504-510, 2006, describing RNAi; Dillon et al., Annual Review of Physiology, 67:147-173, 2005, describing RNAi; Dykxhoorn and Lieberman, Annual Review of Medicine, 56:401-423, 2005, describing RNAi). RNAi molecules are commercially available from a variety of sources (e.g., Ambion, TX; Sigma Aldrich, MO; Invitrogen). Several siRNA molecule design programs using a variety of algorithms are known to the art (see, e.g., Cenix algorithm, Ambion; BLOCK-iT™ RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tools, Bioinformatics & Research Computing). Traits influential in defining optimal siRNA sequences include G/C content at the termini of the siRNAs, Tm of specific internal domains of the siRNA, siRNA length, position of the target sequence within the CDS (coding region), and nucleotide content of the 3′ overhangs.

Breeding

It is disclosed herein that a quantitative trait locus (QTL) with major effects for SCN resistance and single nucleotide polymorphism (SNP) markers in the proximity of this locus have been identified that can be used for the introgression of this genomic region to desirable germplasm, such as by marker-assisted selection and/or marker-assisted backcrossing.

The present disclosure provides genetic markers and methods for the introduction of Glyma18g02570, Glyma18g02580, or Glyma18g02590 alleles into agronomically elite soybean plants. The invention therefore allows the creation of plants that combine these Glyma18g02570, Glyma18g02580, or Glyma18g02590 alleles that confer SCN resistance with a commercially significant yield and an agronomically elite genetic background. Using the methods of the invention, loci conferring the SCN phenotype may be introduced into a desired soybean genetic background, for example, in the production of new varieties with commercially significant yield and SCN resistance.

As used herein, the term “population” means a genetically heterogenous collection of plants that share a common parental derivation.

As used herein, the terms “variety” and “cultivar” mean a group of similar plants that by their genetic pedigrees and performance can be identified from other varieties within the same species.

As used herein, an “allele” refers to one of two or more alternative forms of a genomic sequence at a given locus on a chromosome.

A “Quantitative Trait Locus (QTL)” is a chromosomal location that encodes for alleles that affect the expressivity of a phenotype.

As used herein, a “marker” means a detectable characteristic that can be used to discriminate between organisms. Examples of such characteristics include, but are not limited to, genetic markers, biochemical markers, metabolites, morphological characteristics, and agronomic characteristics.

As used herein, the term “phenotype” means the detectable characteristics of a cell or organism that can be influenced by gene expression.

As used herein, the term “genotype” means the specific allelic makeup of a plant.

“Agronomically elite” refers to a genotype that has a culmination of many distinguishable traits such as emergence, vigor, vegetative vigor, disease resistance, seed set, standability, and threshability, which allows a producer to harvest a product of commercial significance.

As used herein, the term “introgressed,” when used in reference to a genetic locus, refers to a genetic locus that has been introduced into a new genetic background. Introgression of a genetic locus can thus be achieved through plant breeding methods and/or by molecular genetic methods. Such molecular genetic methods include, but are not limited to, various plant transformation techniques and/or methods that provide for homologous recombination, non-homologous recombination, site-specific recombination, and/or genomic modifications that provide for locus substitution or locus conversion.

As used herein, the term “linked,” when used in the context of nucleic acid markers and/or genomic regions, means that the markers and/or genomic regions are located on the same linkage group or chromosome.

As used herein, the term “denoting” when used in reference to a plant genotype refers to any method whereby a plant is indicated to have a certain genotype. This includes any means of identification of a plant having a certain genotype. Indication of a certain genotype may include, but is not limited to, any entry into any type of written or electronic medium or database whereby the plant's genotype is provided. Indications of a certain genotype may also include, but are not limited to, any method where a plant is physically marked or tagged. Illustrative examples of physical marking or tags useful in the invention include, but are not limited to, a barcode, a radio-frequency identification (RFID), a label, or the like.

Marker assisted introgression involves the transfer of a chromosome region defined by one or more markers from one germplasm to a second germplasm. The initial step in that process is the localization of the trait by gene mapping, which is the process of determining the position of a gene relative to other genes and genetic markers through linkage analysis. The basic principle for linkage mapping is that the closer together two genes are on the chromosome, the more likely they are to be inherited together. Briefly, a cross is generally made between two genetically compatible but divergent parents relative to traits under study. Genetic markers can then be used to follow the segregation of traits under study in the progeny from the cross, often a backcross (BC1), F₂, or recombinant inbred population.

The term quantitative trait loci, or QTL, is used to describe regions of a genome showing quantitative or additive effects upon a phenotype. The Rhg1 loci, containing Glyma18g02570, Glyma18g02580, or Glyma18g02590 alleles, represent exemplary QTL because Glyma18g02570, Glyma18g02580, or Glyma18g02590 alleles result in SCN resistance. Herein identified are genetic markers for non-transgenic, Glyma18g02570, Glyma18g02580, or Glyma18g02590 alleles that enable breeding of soybean plants comprising the Glyma18g02570, Glyma18g02580, or Glyma18g02590 alleles with agronomically superior plants, and selection of progeny that inherited the mutant Glyma18g02570, Glyma18g02580, or Glyma18g02590 alleles. Thus, the invention allows the use of molecular tools to combine these QTLs with desired agronomic characteristics.

Various embodiments of the present disclosure utilize a QTL or polymorphic nucleic acid marker or allele located in this genomic region. Subregions of this genomic region associated with SCN resistant phenotype can be described as being flanked by markers shown in TABLE 6. Such markers are believed to be associated with the SCN resistant phenotype because of their location and proximity to the major QTL. One or more polymorphic nucleic acids can be used from TABLE 6. For example, at least two, three, four, five, six, seven, eight, nine, ten, or more of such markers can used.

It can be useful to detect in, or determine whether, a soybean plant has an allelic state that is associated with or not associated with an SCN resistant phenotype.

A plant can be identified in which at least one allele at a polymorphic locus associated with an SCN resistant phenotype is detected. For example, a diploid plant in which the allelic state at a polymorphic locus comprises one allele associated with an SCN resistant phenotype and one allele that is not associated with an SCN resistant phenotype (i.e., heterozygous at that locus). In certain embodiments of the invention, it may be useful to cross a plant that is heterozygous at a locus associated with an SCN resistant phenotype with a plant that is similarly heterozygous or that does not contain any allele associated with an SCN resistant phenotype at the locus, to produce progeny a certain percentage of plants that are heterozygous at that locus. Plants homozygous at the locus may then be produced by various breeding methods, such as by self-crossing or dihaploidization.

One of skill in the art will also recognize that it can be useful to identify at a genetic locus a polymorphic nucleic acid marker that is not associated with an SCN resistant phenotype in a plant, such as when introgressing a QTL associated with an SCN resistant phenotype into a genetic background not associated with such a phenotype.

Markers and allelic states disclosed herein are exemplary. From Table 6, one of skill in the art would recognize how to identify soybean plants with other polymorphic nucleic acid markers and allelic states thereof related to SCN resistance consistent with the present disclosure. One of skill the art would also know how to identify the allelic state of other polymorphic nucleic acid markers located in the genomic region(s) or linked to the QTL or other markers identified herein, to determine their association with SCN resistance.

Provided herein are unique soybean germplasms or soybean plants comprising an introgressed genomic region that is associated with an SCR resistant phenotype and method of obtaining the same. Marker-assisted introgression involves the transfer of a chromosomal region, defined by one or more markers, from one germplasm to a second germplasm. Offspring of a cross that contain the introgressed genomic region can be identified by the combination of markers characteristic of the desired introgressed genomic region from a first germplasm (e.g., an SCN resistant phenotype germplasm) and both linked and unlinked markers characteristic of the desired genetic background of a second germplasm. Flanking markers that identify a genomic region associated with an SCN resistant phenotype include those in TABLE 6.

Flanking markers that fall on both the telomere proximal end and the centromere proximal end of any of these genomic intervals may be useful in a variety of breeding efforts that include, but are not limited to, introgression of genomic regions associated with an SCN resistant phenotype into a genetic background comprising markers associated with germplasm that ordinarily contains a genotype associated with a non-SCN resistant phenotype. Markers that are linked and either immediately adjacent or adjacent to the identified SCN resistant phenotype QTL that permit introgression of the QTL in the absence of extraneous linked DNA from the source germplasm containing the QTL are provided herewith. Those of skill in the art will appreciate that when seeking to introgress a smaller genomic region comprising a QTL associated with an SCN resistant phenotype described herein, that any of the telomere proximal or centromere proximal markers that are immediately adjacent to a larger genomic region comprising the QTL can be used to introgress that smaller genomic region.

Soybean plants or germplasm comprising an introgressed region that is associated with an SCN resistant phenotype wherein at least 10%, 25%, 50%, 75%, 90%, or 99% of the remaining genomic sequences carry markers characteristic of plant or germplasm that otherwise or ordinarily comprise a genomic region associated with an non-SCN resistant phenotype, are thus provided. Furthermore, soybean plants comprising an introgressed region where closely linked regions adjacent or immediately adjacent to the genomic regions, QTL, and markers provided herewith that comprise genomic sequences carrying markers characteristic of soybean plants or germplasm that otherwise or ordinarily comprise a genomic region associated with the phenotype are also provided.

Genetic markers that can be used in the practice of the present disclosure include, but are not limited to, Restriction Fragment Length Polymorphisms (RFLP), Amplified Fragment Length Polymorphisms (AFLP), Simple Sequence Repeats (SSR), Single Nucleotide Polymorphisms (SNP), Insertion/Deletion Polymorphisms (Indels), Variable Number Tandem Repeats (VNTR), and Random Amplified Polymorphic DNA (RAPD), and others known to those skilled in the art. Marker discovery and development in crops provides the initial framework for applications to marker-assisted breeding activities (U.S. Patent Pub. Nos.: 2005/0204780, 2005/0216545, 2005/0218305, and 2006/00504538). The resulting “genetic map” is the representation of the relative position of characterized loci (polymorphic nucleic acid markers or any other locus for which alleles can be identified) to each other.

As a set, polymorphic markers serve as a useful tool for fingerprinting plants to inform the degree of identity of lines or varieties (U.S. Pat. No. 6,207,367). These markers form the basis for determining associations with phenotypes and can be used to drive genetic gain. In certain embodiments of the present disclosure, polymorphic nucleic acids can be used to detect in a soybean plant a genotype associated with an SCN resistant phenotype, identify a soybean plant with a genotype associated with an SCN resistant phenotype, or to select a soybean plant with a genotype associated with an SCN resistant phenotype. In certain embodiments of methods of the present disclosure, polymorphic nucleic acids can be used to produce a soybean plant that comprises in its genome an introgressed locus associated with an SCN resistant phenotype. In certain embodiments of the invention, polymorphic nucleic acids can be used to breed progeny soybean plants comprising a locus associated with an SCN resistant phenotype.

Certain genetic markers useful in the present invention include “dominant” or “codominant” markers. “Codominant” markers reveal the presence of two or more alleles (two per diploid individual). “Dominant” markers reveal the presence of only a single allele. The presence of the dominant marker phenotype (e.g., a band of DNA) is an indication that one allele is present in either the homozygous or heterozygous condition. The absence of the dominant marker phenotype (e.g., absence of a DNA band) is merely evidence that “some other” undefined allele is present. In the case of populations where individuals are predominantly homozygous and loci are predominantly dimorphic, dominant and codominant markers can be equally valuable. As populations become more heterozygous and multiallelic, codominant markers often become more informative of the genotype than dominant markers.

Nucleic acid-based analyses for determining the presence or absence of the genetic polymorphism (i.e. for genotyping) can be used in breeding programs for identification, selection, introgression, or the like. A wide variety of genetic markers for the analysis of genetic polymorphisms are available and known to those of skill in the art. The analysis may be used to select for genes, portions of genes, QTL, alleles, or genomic regions that comprise or are linked to a genetic marker that is linked to or associated with an SCN resistant phenotype.

As used herein, nucleic acid analysis methods include, but are not limited to, PCR-based detection methods (for example, TaqMan assays), microarray methods, mass spectrometry-based methods and/or nucleic acid sequencing methods, including whole genome sequencing. In certain embodiments, the detection of polymorphic sites in a sample of DNA, RNA, or cDNA may be facilitated through the use of nucleic acid amplification methods. Such methods specifically increase the concentration of polynucleotides that span the polymorphic site, or include that site and sequences located either distal or proximal to it. Such amplified molecules can be readily detected by gel electrophoresis, fluorescence detection methods, or other means.

One method of achieving such amplification employs the polymerase chain reaction (PCR) (Mullis et al. 1986 Cold Spring Harbor Symp. Quant. Biol. 51:263-273; European Patent 50,424; European Patent 84,796; European Patent 258,017; European Patent 237,362; European Patent 201,184; U.S. Pat. No. 4,683,202; U.S. Pat. No. 4,582,788; and U.S. Pat. No. 4,683,194), using primer pairs that are capable of hybridizing to the proximal sequences that define a polymorphism in its double-stranded form. Methods for typing DNA based on mass spectrometry can also be used. Such methods are disclosed in U.S. Pat. Nos. 6,613,509 and 6,503,710, and references found therein.

Polymorphisms in DNA sequences can be detected or typed by a variety of effective methods well known in the art including, but not limited to, those disclosed in U.S. Pat. Nos. 5,468,613, 5,217,863; 5,210,015; 5,876,930; 6,030,787; 6,004,744; 6,013,431; 5,595,890; 5,762,876; 5,945,283; 5,468,613; 6,090,558; 5,800,944; 5,616,464; 7,312,039; 7,238,476; 7,297,485; 7,282,355; 7,270,981 and 7,250,252 all of which are incorporated herein by reference in their entireties. However, the compositions and methods of the present disclosure can be used in conjunction with any polymorphism typing method to type polymorphisms in genomic DNA samples. These genomic DNA samples used include but are not limited to genomic DNA isolated directly from a plant, cloned genomic DNA, or amplified genomic DNA.

For example, polymorphisms in DNA sequences can be detected by hybridization to allele-specific oligonucleotide (ASO) probes as disclosed in U.S. Pat. Nos. 5,468,613 and 5,217,863. U.S. Pat. No. 5,468,613 discloses allele specific oligonucleotide hybridizations where single or multiple nucleotide variations in nucleic acid sequence can be detected in nucleic acids by a process in which the sequence containing the nucleotide variation is amplified, spotted on a membrane and treated with a labeled sequence-specific oligonucleotide probe.

Target nucleic acid sequence can also be detected by probe ligation methods as disclosed in U.S. Pat. No. 5,800,944 where sequence of interest is amplified and hybridized to probes followed by ligation to detect a labeled part of the probe.

Microarrays can also be used for polymorphism detection, wherein oligonucleotide probe sets are assembled in an overlapping fashion to represent a single sequence such that a difference in the target sequence at one point would result in partial probe hybridization (Borevitz et al., Genome Res. 13:513-523 (2003); Cui et al., Bioinformatics 21:3852-3858 (2005). On any one microarray, it is expected there will be a plurality of target sequences, which may represent genes or non-coding regions wherein each target sequence is represented by a series of overlapping oligonucleotides, rather than by a single probe. This platform provides for high throughput screening of a plurality of polymorphisms. Typing of target sequences by microarray-based methods is disclosed in U.S. Pat. Nos. 6,799,122; 6,913,879; and 6,996,476.

Target nucleic acid sequence can also be detected by probe linking methods as disclosed in U.S. Pat. No. 5,616,464, employing at least one pair of probes having sequences homologous to adjacent portions of the target nucleic acid sequence and having side chains which non-covalently bind to form a stem upon base pairing of the probes to the target nucleic acid sequence. At least one of the side chains has a photoactivatable group which can form a covalent cross-link with the other side chain member of the stem.

Other methods for detecting SNPs and Indels include single base extension (SBE) methods. Examples of SBE methods include, but are not limited, to those disclosed in U.S. Pat. Nos. 6,004,744; 6,013,431; 5,595,890; 5,762,876; and 5,945,283. SBE methods are based on extension of a nucleotide primer that is adjacent to a polymorphism to incorporate a detectable nucleotide residue upon extension of the primer. In certain embodiments, the SBE method uses three synthetic oligonucleotides. Two of the oligonucleotides serve as PCR primers and are complementary to sequence of the locus of genomic DNA which flanks a region containing the polymorphism to be assayed. Following amplification of the region of the genome containing the polymorphism, the PCR product is mixed with the third oligonucleotide (called an extension primer) which is designed to hybridize to the amplified DNA adjacent to the polymorphism in the presence of DNA polymerase and two differentially labeled dideoxynucleosidetriphosphates. If the polymorphism is present on the template, one of the labeled dideoxynucleosidetriphosphates can be added to the primer in a single base chain extension. The allele present is then inferred by determining which of the two differential labels was added to the extension primer. Homozygous samples will result in only one of the two labeled bases being incorporated and thus only one of the two labels will be detected. Heterozygous samples have both alleles present, and will thus direct incorporation of both labels (into different molecules of the extension primer) and thus both labels will be detected.

In another method for detecting polymorphisms, SNPs and Indels can be detected by methods disclosed in U.S. Pat. Nos. 5,210,015; 5,876,930; and 6,030,787 in which an oligonucleotide probe having a 5′ fluorescent reporter dye and a 3′ quencher dye covalently linked to the 5′ and 3′ ends of the probe. When the probe is intact, the proximity of the reporter dye to the quencher dye results in the suppression of the reporter dye fluorescence, e.g. by Forster-type energy transfer. During PCR forward and reverse primers hybridize to a specific sequence of the target DNA flanking a polymorphism while the hybridization probe hybridizes to polymorphism-containing sequence within the amplified PCR product. In the subsequent PCR cycle DNA polymerase with 5′ to 3′ exonuclease activity cleaves the probe and separates the reporter dye from the quencher dye resulting in increased fluorescence of the reporter.

In another embodiment, the locus or loci of interest can be directly sequenced using nucleic acid sequencing technologies. Methods for nucleic acid sequencing are known in the art and include technologies provided by 454 Life Sciences (Branford, Conn.), Agencourt Bioscience (Beverly, Mass.), Applied Biosystems (Foster City, Calif.), LI-COR Biosciences (Lincoln, Nebr.), NimbleGen Systems (Madison, Wis.), Illumina (San Diego, Calif.), and VisiGen Biotechnologies (Houston, Tex.). Such nucleic acid sequencing technologies comprise formats such as parallel bead arrays, sequencing by ligation, capillary electrophoresis, electronic microchips, “biochips,” microarrays, parallel microchips, and single-molecule arrays, as reviewed by R.F. Service Science 2006 311:1544-1546.

The markers to be used in the methods of the present disclosure can be diagnostic of origin in order for inferences to be made about subsequent populations. Experience to date suggests that SNP markers may be ideal for mapping because the likelihood that a particular SNP allele is derived from independent origins in the extant populations of a particular species is very low. As such, SNP markers (see e.g., TABLE 6) appear to be useful for tracking and assisting introgression of QTLs.

Research Tools

The Glyma18g02570, Glyma18g02580, or Glyma18g02590 genes can be used to find or characterize related (interactive) genes or identify or further characterize the cascade for SCN resistance. The discovery of a Glyma18g02570, Glyma18g02580, or Glyma18g02590 as part of the resistance signaling pathway against SCN provides novel insight into this complex host-pathogen interaction. Insights reported herein can be used to discern the relationship between Glyma18g02570, Glyma18g02580, or Glyma18g02590 and metabolism.

In some embodiments, the Glyma18g02570, Glyma18g02580, or Glyma18g02590 genes can be used in a genomics, proteomics, bioinformatics, or statistical modeling approach to fish or isolate candidate genes or encoded proteins or other molecules with a direct or indirect function in mediating disease resistance to SCN in soybeans. In some embodiments, the Glyma18g02570, Glyma18g02580, or Glyma18g02590 genes can be used in a genomics, proteomics, bioinformatics, or statistical modeling approach to fish or isolate candidate genes or encoded proteins or other molecules with a direct or indirect function in mediating compatible or incompatible responses of soybeans to SCN (e.g., to a nematode or any intermediate). Thus is provided various methods to find or characterize related (interactive) genes involved with SCN resistance.

Targeting-Induced Local Lesions in Genomes (TILLING) is a method permitting identification of gene-specific mutations. In particular, this process uses traditional mutagenesis and SNP discovery methods for a reverse genetic strategy that takes advantage of a mismatch endonuclease to locate and detect induced mutations in a high-throughput and low cost manner. EcoTILLING, which is a variant of TILLING, examines natural genetic variation in populations to discover SNPs (reviewed in Barkley and Wang, Curr Genomics, 9(4):212-26, 2008).

Kits

Also provided are kits. Such kits can include an agent or composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Components include, but are not limited to an antibody (e.g., a monoclonal antibody) specific for a transcribable nucleic acid molecule described herein (e.g., SEQ ID NOS: 1-3, or variants thereof) or encoded polypeptides disclosed herein (e.g., SEQ ID NOS: 4-6, or variants thereof). Methods for generating such a monoclonal antibody are known in the art and can be adapted to the methods or compositions described herein. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components.

Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline or sterile each of which has been packaged under a neutral non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.

In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic-readable medium, such as a floppy disc, mini-CD-ROM, CD-ROM, DVD-ROM, Zip disc, videotape, audio tape, and the like. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit.

Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.

EXAMPLES

The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

Example 1 Positional Cloning of the Rhg1 Gene

The following example describes the positional cloning of the Rhg1 gene. Three genetic populations segregating for resistance to SCN PA3 (Hgtype 0) were used for mapping. These included an F2:6 recombinant inbred line (RIL) population from a cross between Forrest and Essex (98 individuals; Meksem et al., 2001), and two large F2 populations generated from crosses between Forrest and either Essex (1,755 lines) or Williams 82 (2,060 lines).

To enrich the chromosomal interval carrying the Rhg1 locus with recombinants, SCN phenotyping was conducted according to Brown et al. (2010). Because Forrest SCN-resistance requires both the Rhg1 and Rhg4 loci (Meksem et al., 2001), genotyping was conducted using DNA markers flanking both loci to detect informative recombinants at the Rhg1 locus. The SSR markers, Sat_(—)210 and Satt309 (see, e.g., SoyBase and the Soybean Breeder's Toolbox at soybase.org), and SIUC-SAT143 were used to identify chromosomal breakpoints at the Rhg1 locus and the Rhg1 genotype of each recombinant. PCR amplifications were performed using DNA from individuals from each of the three genetic populations. To enrich the chromosomal regions carrying the Rhg1 locus with DNA markers, the GenBank published Williams 82 sequences were used to design PCR primers every 5 to 10 kbp of the 370 kbp carrying the Rhg1 locus. DNA from Forrest and Essex were tested with each primer using a modified EcoTILLING protocol to find and map polymorphic sequences at the Rhg1 locus (Meksem et al., 2008; Liu et al., 2011). The identified SNP and InDel DNA markers were integrated into the informative recombinants to identify chromosomal breakpoints and the interval that carried the Rhg1 locus. A high density genetic map was developed for the Rhg1 locus (see e.g., FIG. 1). Comparison of the SNAP gene sequences between Forrest and Essex identified some significant changes including three SNPs (G2464C, T4206G and A4215C) and three InDels (G4211-, G4212- and G4213-) within the exons (see e.g., FIG. 1B).

Example 2 Relationship Between Genes and Resistance to SCN

The following example shows the relationship between the Glyma18g02570 (armadillo/beta-catenin-like repeat), Glyma18g02580 (amino acid transporter) and Glyma18g02590 (SNAP) genes and resistance to SCN. A haplotype map was developed using 4 DNA markers (560, 570, 590 and Satt309) at the Rhg1 locus and 1 DNA marker (Sat_(—)162) plus the Rhg4 GmSHMT gene at the Rhg4 locus, respectively The Forrest genotype was classified resistant (R) and the Essex genotype was classified susceptible (S). Lines were classified resistant (R) to SCN if female index (FI)≦10% and susceptible (S) if FI>10% (see e.g., TABLE 2).

TABLE 2 Haplotype map of SCN resistance in soybean. SCN infection Rhg1 locus Rhg4 locus Plant line phenotype 560 570 590 Satt309 GmSHMT Sat_162 Forrest R R R R R R R Peking R R R R R R R PI437654 R R R R R R R PI89772 R R R R R R R PI90763 R R R R R R R PI88788 R R R R S S R PI546316 R R R R S S R PI209332 R R R R S S S Essex S S S S S S S Williams 82 S S S S S S S PI603428C S S S S R S R

In addition, a detailed haplotype analysis was conducted for the SNAP gene. The SNAP coding region from 11 soybean lines was sequenced, representing the SCN-resistance variability in soybean germplasm. The amino acid differences in the predicted protein sequences of SNAP from the 11 soybean lines are shown with the number indicating the amino acid position in the predicted protein (see e.g., FIG. 2B). Haplotyping results from these 11 soybean lines indicate three types of SNAP haplotypes. The data further indicates there are at least two resistant types: Peking Type I including Peking, Forrest, PI437654, PI89772 and PI90763, and PI88788 Type II including PI88788, PI548316 and PI209332; and one susceptible Type III including Essex, Williams 82 and PI603428C.

Example 3 Virus-Induced Gene-Silencing (VIGS)

The following example describes VIGS in soybean. Bean pod mottle virus (BPMV) VIGS vectors, pBPMV IA-R1M, and pBPMV-IA-D35 were used in this example (Zhang et. al., 2010). pBPMV-IA-D35 is a derivative of pBPMV-IA-R2 containing BamHI and KpnI restriction sites between the cistrons encoding the movement protein and the large coat protein 15 subunit. Briefly, a 328 bp fragment of the SNAP cDNA sequence was amplified from soybean (cv. Forrest) root cDNA by RT-PCR. PCR products were digested with BamHI and KpnI and ligated into pBPMV-IA-D35 digested with the same enzymes to generate pBPMV-IA-SNAP. Gold particles coated with plasmid DNA corresponding to pBPMV-IA-R1M and pBPMV-IA-SNAP were co-bombarded into soybean leaf tissue (Zhang et al., 2010). At 3-4 weeks post-inoculation, BPMV-infected leaves were collected, lyophilized, and stored at −20° C. for future experiments. Infected soybean leaf tissues were ground with a mortar and pestle in 0.05 M potassium phosphate buffer (pH 7.0) and used as virus inoculum for VIGS assays.

The SCN-resistant RIL ExF67 was inoculated with pBPMV-IA-SNAP (Glyma18g02590). Control plants were infected with BPMV only. Each treatment consisted of at least 12 plants. Unifoliate leaves of 9-day-old plants were rub-inoculated with virus using carborundum (Zhang et al., 2010). Plants were grown in a growth chamber set to the following conditions: 20-21° C., 16 h light/8 h dark, and 100 mE m-2s-1 light intensity. A strong hypersensitive cell death-like response was observed in the leaves of infected pBPMV-IA-SNAP plants (see e.g., FIG. 3) and also resulted in poor root development that compromised the ability to conduct nematode infection assays on these plants.

Plants silenced for Glyma18g02590.1 in soybean leaves caused a strong hypersensitive cell death response (necrotic lesions) and compromised root growth. Consequently, the plants were not phenotyped against SCN (see e.g., FIG. 3).

SCN-resistance can be manifested at the site of nematode feeding as a strong hypersensitive response (HR) that leads to death of the feeding cell and nematode. Thus, these data indicate that interference in SNAP gene function in the resistant cultivar can mediate the SCN-resistance response.

Example 4 Near Isogenic Lines (NILs)

The following example describes additional evidence from Near Isogenic Lines (NILs). NILs that differ in SCN-resistance because of variations at the Rhg1 locus, but not the Rhg4 locus, were analyzed by genome resequencing and GoldenGate SNP analysis. SNPs in and around the three additional genes were found to be polymorphic (see e.g., TABLE 3) and therefore were identified as conferring SCN-resistance.

TABLE 3 NIL polymorphisms around the three additional genes underlying SCN-resistance. EXF34- EXF34-32 Index Name Position 23Sus Res 43751 Gm18_1552671_A_G 1552671 BB AA 43760 Gm18_1562162_G_A 1562162 AA BB 43771 Gm18_1567581_G_A 1567581 BB NC 43784 Gm18_1582570_T_C 1582570 BB BB 43815 Gm18_1612017_G_A 1612017 BB AA 43823 Gm18_1620585_T_C 1620585 BB BB 43829 Gm18_1625693_A_G 1625693 BB BB 43836 Gm18_1630870_C_A 1630870 BB BB 43840 Gm18_1634453_G_A 1634453 AA AA 43845 Gm18_1640404_C_A 1640404 BB AA 43849 Gm18_1652357_C_T 1652357 BB AA 43859 Gm18_1663298_A_G 1663298 BB BB 43863 Gm18_1671483_A_G 1671483 AA BB 43865 Gm18_1674972_C_T 1674972 AA AA 43869 Gm18_1677273_T_G 1677273 NC BB

Example 5 Sequencing Recombinant Inbred Lines

The following example describes additional evidence from sequencing recombinant Inbred lines (RILs). RILs that differ in SCN-resistance because of variations at the Rhg1 locus, but not the Rhg4 locus, were analyzed by genome resequencing. SNPs in and around the three additional genes were found to be polymorphic (see e.g., TABLE 4) and therefore conferring SCN-resistance.

TABLE 4 SNPs among RILs from Illumina Sequencing. >SNP_Gm18_15187890 Essex: A Forrest: G 1 SNP A 17 17 0.000008 PARENT_A Essex 2 COV G 31 31 0.000000 PARENT_B Forrest 3 SNP A 19 19 0.000002 PARENT_A RIL-1 4 COV G 12 12 0.000244 PARENT_B RIL-10 5 COV G 19 19 0.000002 PARENT_B RIL-11 6 SNP A 33 33 0.000000 PARENT_A RIL-12 7 SNP A 15 15 0.000031 PARENT_A RIL-13 9 COV G 30 37 0.000075 PARENT_B RIL-15 10 SNP A 13 13 0.000122 PARENT_A RIL-17 11 SNP A 25 25 0.000000 PARENT_A RIL-18 12 SNP A 19 19 0.000002 PARENT_A RIL-19 13 COV G 8 9 0.017578 PARENT_B RIL-2 14 COV G 11 11 0.000488 PARENT_B RIL-20 16 COV G 24 24 0.000000 PARENT_B RIL-22 17 COV G 21 21 0.000000 PARENT_B RIL-23 18 COV G 18 18 0.000004 PARENT_B RIL-24 19 COV G 10 10 0.000977 PARENT_B RIL-25 20 SNP A 8 8 0.003906 PARENT_A RIL-26 21 COV G 33 33 0.000000 PARENT_B RIL-27 22 COV G 19 19 0.000002 PARENT_B RIL-28 24 COV G 15 15 0.000031 PARENT_B RIL-37 25 SNP A 6 6 0.015625 PARENT_A RIL-38 26 SNP A 8 8 0.003906 PARENT_A RIL-4 27 SNP A 28 28 0.000000 PARENT_A RIL-40 28 COV G 18 18 0.000004 PARENT_B RIL-41 29 COV G 23 23 0.000000 PARENT_B RIL-42 30 COV G 22 25 0.000069 PARENT_B RIL-44 31 COV G 19 26 0.009802 PARENT_B RIL-46 32 SNP A 28 28 0.000000 PARENT_A RIL-47 34 COV G 9 9 0.001953 PARENT_B RIL-49 35 SNP A 15 15 0.000031 PARENT_A RIL-51 37 SNP A 53 53 0.000000 PARENT_A RIL-53 38 COV G 39 39 0.000000 PARENT_B RIL-54 39 COV G 22 25 0.000069 PARENT_B RIL-55 40 COV G 26 26 0.000000 PARENT_B RIL-56 41 COV G 25 25 0.000000 PARENT_B RIL-57 42 COV G 34 34 0.000000 PARENT_B RIL-58 43 SNP A 6 6 0.015625 PARENT_A RIL-6 44 SNP A 27 27 0.000000 PARENT_A RIL-67 45 SNP A 73 73 0.000000 PARENT_A RIL-7 46 SNP A 17 17 0.000008 PARENT_A RIL-72 47 COV G 20 25 0.001583 PARENT_B RIL-74 48 SNP A 31 31 0.000000 PARENT_A RIL-75 50 COV G 23 23 0.000000 PARENT_B RIL-79 51 COV G 40 40 0.000000 PARENT_B RIL-8 52 SNP A 43 43 0.000000 PARENT_A RIL-80 53 COV G 53 53 0.000000 PARENT_B RIL-84 54 SNP A 26 26 0.000000 PARENT_A RIL-85 55 COV G 40 40 0.000000 PARENT_B RIL-89 56 SNP A 47 47 0.000000 PARENT_A RIL-9 57 COV G 26 26 0.000000 PARENT_B RIL-91 58 COV G 34 34 0.000000 PARENT_B RIL-92 59 COV G 28 28 0.000000 PARENT_B RIL-94 60 COV G 27 27 0.000000 PARENT_B RIL-95 61 SNP A 37 37 0.000000 PARENT_A RIL-96 >SNP_Gm18_1608702 Essex: A Forrest: T 1 SNP A 58 58 0.000000 PARENT_A Essex 2 COV T 114 114 0.000000 PARENT_B Forrest 3 COV T 58 58 0.000000 PARENT_B RIL-1 4 COV T 42 65 0.006155 PARENT_B RIL-10 5 COV T 38 44 0.000000 PARENT_B RIL-11 6 COV T 70 70 0.000000 PARENT_B RIL-12 7 COV T 44 46 0.000000 PARENT_B RIL-13 8 SNP A 47 47 0.000000 PARENT_A RIL-14 9 COV T 41 72 0.047140 PARENT_B RIL-15 10 COV T 51 58 0.000000 PARENT_B RIL-17 11 SNP A 46 46 0.000000 PARENT_A RIL-18 12 SNP A 52 52 0.000000 PARENT_A RIL-19 13 COV T 27 27 0.000000 PARENT_B RIL-2 14 COV T 30 30 0.000000 PARENT_B RIL-20 15 COV T 65 65 0.000000 PARENT_B RIL-21 16 SNP A 77 77 0.000000 PARENT_A RIL-22 17 COV T 30 30 0.000000 PARENT_B RIL-23 18 COV T 53 53 0.000000 PARENT_B RIL-24 19 COV T 30 30 0.000000 PARENT_B RIL-25 20 COV T 28 36 0.000440 PARENT_B RIL-26 21 SNP A 111 111 0.000000 PARENT_A RIL-27 22 SNP A 23 23 0.000000 PARENT_A RIL-28 23 COV T 29 29 0.000000 PARENT_B RIL-36 24 COV T 25 25 0.000000 PARENT_B RIL-37 25 COV T 37 37 0.000000 PARENT_B RIL-38 26 COV T 32 41 0.000159 PARENT_B RIL-4 27 COV T 72 72 0.000000 PARENT_B RIL-40 28 SNP A 49 49 0.000000 PARENT_A RIL-41 29 SNP A 51 51 0.000000 PARENT_A RIL-42 30 COV T 57 57 0.000000 PARENT_B RIL-44 32 COV T 47 47 0.000000 PARENT_B RIL-47 33 SNP A 18 18 0.000004 PARENT_A RIL-48 34 SNP A 40 40 0.000000 PARENT_A RIL-49 35 SNP A 24 24 0.000000 PARENT_A RIL-51 36 SNP A 87 87 0.000000 PARENT_A RIL-52 37 SNP A 99 99 0.000000 PARENT_A RIL-53 38 SNP A 88 91 0.000000 PARENT_A RIL-54 39 COV T 28 43 0.017227 PARENT_B RIL-55 40 COV T 59 59 0.000000 PARENT_B RIL-56 41 COV T 79 80 0.000000 PARENT_B RIL-57 43 SNP A 27 27 0.000000 PARENT_A RIL-6 44 COV T 91 91 0.000000 PARENT_B RIL-67 45 COV T 148 148 0.000000 PARENT_B RIL-7 46 SNP A 50 50 0.000000 PARENT_A RIL-72 47 COV T 65 65 0.000000 PARENT_B RIL-74 48 COV T 74 83 0.000000 PARENT_B RIL-75 49 SNP A 124 124 0.000000 PARENT_A RIL-76 50 SNP A 78 78 0.000000 PARENT_A RIL-79 51 SNP A 83 83 0.000000 PARENT_A RIL-8 52 SNP A 103 103 0.000000 PARENT_A RIL-80 53 COV T 102 102 0.000000 PARENT_B RIL-84 54 SNP A 70 70 0.000000 PARENT_A RIL-85 55 COV T 121 121 0.000000 PARENT_B RIL-89 56 SNP A 123 123 0.000000 PARENT_A RIL-9 57 SNP A 73 73 0.000000 PARENT_A RIL-91 58 SNP A 81 81 0.000000 PARENT_A RIL-92 59 SNP A 79 79 0.000000 PARENT_A RIL-94 60 COV T 48 49 0.000000 PARENT_B RIL-95 61 SNP A 124 124 0.000000 PARENT_A RIL-96 >SNP_Gm18_1608832 Essex: G Forrest: A 1 SNP G 5 5 0.031250 PARENT_A Essex 2 COV A 9 9 0.001953 PARENT_B Forrest 3 COV A 14 14 0.000061 PARENT_B RIL-1 4 COV A 17 19 0.000326 PARENT_B RIL-10 5 COV A 9 10 0.009766 PARENT_B RIL-11 6 COV A 31 31 0.000000 PARENT_B RIL-12 7 COV A 11 12 0.002930 PARENT_B RIL-13 8 SNP G 11 11 0.000488 PARENT_A RIL-14 10 COV A 21 22 0.000005 PARENT_B RIL-17 11 SNP G 18 18 0.000004 PARENT_A RIL-18 12 SNP G 5 5 0.031250 PARENT_A RIL-19 14 COV A 11 11 0.000488 PARENT_B RIL-20 15 COV A 19 19 0.000002 PARENT_B RIL-21 16 SNP G 20 20 0.000001 PARENT_A RIL-22 17 COV A 10 10 0.000977 PARENT_B RIL-23 18 COV A 20 20 0.000001 PARENT_B RIL-24 19 COV A 11 11 0.000488 PARENT_B RIL-25 21 SNP G 31 31 0.000000 PARENT_A RIL-27 22 SNP G 17 17 0.000008 PARENT_A RIL-28 24 COV A 9 9 0.001953 PARENT_B RIL-37 28 SNP G 20 20 0.000001 PARENT_A RIL-41 29 SNP G 16 16 0.000015 PARENT_A RIL-42 30 COV A 11 11 0.000488 PARENT_B RIL-44 32 COV A 15 15 0.000031 PARENT_B RIL-47 33 SNP G 5 5 0.031250 PARENT_A RIL-48 36 SNP G 13 13 0.000122 PARENT_A RIL-52 37 SNP G 38 39 0.000000 PARENT_A RIL-53 38 SNP G 38 40 0.000000 PARENT_A RIL-54 40 COV A 18 18 0.000004 PARENT_B RIL-56 41 COV A 26 26 0.000000 PARENT_B RIL-57 43 SNP G 14 14 0.000061 PARENT_A RIL-6 44 COV A 28 28 0.000000 PARENT_B RIL-67 45 COV A 44 44 0.000000 PARENT_B RIL-7 46 SNP G 24 24 0.000000 PARENT_A RIL-72 47 COV A 11 11 0.000488 PARENT_B RIL-74 48 COV A 18 18 0.000004 PARENT_B RIL-75 49 SNP G 39 39 0.000000 PARENT_A RIL-76 50 SNP G 19 19 0.000002 PARENT_A RIL-79 51 SNP G 28 28 0.000000 PARENT_A RIL-8 52 SNP G 34 34 0.000000 PARENT_A RIL-80 53 COV A 30 30 0.000000 PARENT_B RIL-84 54 SNP G 25 25 0.000000 PARENT_A RIL-85 55 COV A 42 42 0.000000 PARENT_B RIL-89 56 SNP G 29 29 0.000000 PARENT_A RIL-9 57 SNP G 26 26 0.000000 PARENT_A RIL-91 58 SNP G 28 28 0.000000 PARENT_A RIL-92 59 SNP G 30 30 0.000000 PARENT_A RIL-94 61 SNP G 28 28 0.000000 PARENT_A RIL-96

Example 6 Targeted Genome Enrichment and Snap Identification

The following example describes additional evidence for the identification of Rhg1 gene, Glyma18g02590 (SNAP), conferring resistance to SCN, by next-generation sequencing of a targeted 300 kb region of Gm18 in soybean.

TABLE 5 SNPs, insertions, and deletions at the targeted 300 kb region of Gm18 (Gm18: 1480001-1780000) in Essex, Forrest, Peking, and PI88788. Essex Forrest Peking PI88788 Total SNPs 632 618 649 736 1081 Insertions 109 120 123 120 183 Deletions 146 97 100 165 208 Total 887 835 872 1021 1472

TABLE 6 Gm18 SNAP-cluster SNPS. SEQ ID Position Sequence NO: 1552671 AGAG-AGGAA-GGG-AG-AGAAGA--AAAAGA    8 1552732 TGGGGG-G--KGGGGGGGGGTGGTTGGTGTGG    9 1552753 AGAGAAGGA-AGGGGAGGAGAAGAARAAA-GA   10 1552799 CCCCCCAACCCCCC-CCCCCCCCCCCCCCCCC   11 1553174 GAGAGGGGGGGAAAAGAAGGGGGGGGGGGGGG   12 1553377 CTCTCCCTCCCTTTTCTTCTCCTCCYCCCCTC   13 1553485 TCTCTTTTTTTCCCCTCCTTTTTTTTTTTTTT   14 1553949 ACACAAAAAAACCC-ACCAAAAAAAAAAAAAA   15 1554124 GGGGGG-GGGGGGGGGGGGTG-TG--GGGGT-   16 1554570 AAAAAACCAAAAAAA-AAAAAAAA-AAAAAAA   17 1554604 AAAAAAGGAAAAAAA-AARAAAAA-AAA-AAA   18 1554733 T-TWTT-TTTTWWT--T--TTTTA-TTTTTTT   19 1554848 TTTTTTCCTTTTTTT-TTTTTTTTTTTTTTTT   20 1554938 TATAT-AATTTAAAATAA-AT-ATTATTTT-T   21 1554942 AAAAAAAAAA-AAAAAAA-AAAAAAAWAAA-W   22 1555085 ACAC-ACCAAACCCC--CACAACAAM---ACA   23 1555204 AT-TAAT-AAA-TTTATTATAATAA-AAAATA   24 1555236 AAAAAAAAAAA-AAAAA-ATAATAAAAAA-TA   25 1555481 A-A-AA---AA-AA-AAAA-AA-AAWAA--TA   26 1555562 AAA-AAAMAAA-AA----A-A-M-A--AA---   27 1555572 A--TAA-AAAA----AT--AA-A-A--A----   28 1555739 ACACAAA-A-A-CCC-CCAAAAAA-AAAA-A-   29 1555772 G-G-GGG-G-G-TTTG--GGGGGGGGGGG-GG   30 1556277 GAGAGGGGGGGAAAAGAAGAGG-GGRGGGGAG   31 1556372 GAGAGGGGGGGAAA-GAAG-GGGGGGGGGGGG   32 1556588 GCGCGGGGG-GCCCCGCCGGGGGGGGGGGGGG   33 1556678 AA-AGG-GAGRAAAAGAAGAAAAAARGAGAAG   34 1556781 TCTCTTCCTTTCCC-TCCTTTTTTTTTTTTTT   35 1557165 CTTTTTTTCTYTTTTTT-TTCCTTCTTCTCTT   36 1557549 AAAAAWA-A-AAAAAAAAAAAATAAWTA--A-   37 1557751 AGA-AAAAAAA--G-A-GAAAAAAAAAAAAAA   38 1557752 CGC-CCCCCCC--G-C-GCCCCCCCCCCCCCC   39 1557771 TATATTTTTTT--A-TA-TTTTTTTTTTTTTT   40 1557934 GGRGGAGGGGGGGGGGGGAGGGGGGRAGAGGA   41 1557991 TTTTTTTTTTTTTTTTT-TTTTTCTTTTTTCT   42 1558100 AAAAAAAAAAAAAAAAAAATAATTAWAAAA-A   43 1558103 CCTCTTCCCCCCCCCTCCTTCCTTCTTCTC--   44 1558128 GG-GGGG-GTGGGGT-GG--GGGGGG-GGG--   45 1558129 TT-TTTT-TGTTTTG-TT--TTTTTT-TTT--   46 1558137 T----TT-TTT-ATT-----TT-TT--TWT--   47 1558318 AA-ATTA-AAWAAAAT-ATTAATTA--A-AT-   48 1558319 AA-AATA-AAAAAAAA-A-TAATTA--A-AT-   49 1558322 AAAAT-A-AAWAAAAT-A-AAATAA--A-AA-   50 1558323 TTATA-T-TTWTTTTA-T-TTTA-T--T-TT-   51 1558334 TTGTGGT-TTKTTTT--TGGTTGGT--T-TGG   52 1558551 TCTCTTCCTCTCCCCTCCTTTTTTTYTTTTTT   53 1558913 AAAAAGAAAAAAAAAAAAGAAAAAAR-AGAAG   54 1558979 GGAGAGGGGGGGGGG--GGGGGGGGGGGGGGG   55 1559151 ATTTTATTAT-TTTTTTTATAATT-WAAAATA   56 1559399 ATATAA-AAAATTTTATTAAAAAAAAAAAAAA   57 1559585 CACA-CCCCCCAAAACAACCCCCCCC-CCCCC   58 1559603 CCCC-T-CCCCCCCCCCCTCCCCC-TTCTCCT   59 1559659 GAGAGA-AGAGAAAAGAAAAGGAAGAAGAGAA   60 1559787 AAAAACAAAAAAAAAAAACAAAA-AMC-CAAC   61 1559970 TTKTTG-TTTT-TTTTTTGTTTTTTKGTGTTG   62 1560043 TTATAT-TTATTTTTATTTTTTTT-TTTTTTT   63 1560088 CCCCCCTCCTCCCCCCCCCCCCCCCCCCCCCC   64 1560108 TGTGTTTTTTTGGGGTGGTTTTT-TTTTTTTT   65 1560166 GGAGAGAGR-GGGGGAGGG-GGGGGGGGGGG-   66 1560182 TTGTGTGTT-TTTTT-TTTTTTTTTTTTTTTT   67 1560390 AAMACACAA-AAAAAC-AAAAAAAAAAAA-AA   68 1560442 TTTTTTATTATTTTTTTT-TTTTTTTTTTTTT   69 1560517 GARAGAAAGAGAAAAGAAAGGGAA-AAGAGAA   70 1560584 CAMACA-ACCCAAAACAAACCCAA-AACACAA   71 1560705 ACCMCCCCAC-CCCCCCCCAA-CCACCACACC   72 1560784 AAAAAAG--G-AAAAAAAAAAAAAAAA-AAAA   73 1560860 CTCTCTYTCCCTTTTCTTTCCCTTCTTCTCTT   74 1561009 GGGGGGAGGA-GGGGG-GGGGGGGGGGGGGGG   75 1561036 ACCCCCCCACACCCCCCCCAAACCACCACACC   76 1561047 AAGAGAAAAAAAAAAGAAAAAAAA-AAAAAAA   77 1561190 TTTTTTATTATTTT-TTTTTTTTTTTTTTTTT   78 1561230 A-GAGAAAAAAAAAAGAA-AAAAA-AAA-AAA   79 1561392 AA-AAAAAAA-AAAAAAAAAAAGG-GAAAAGA   80 1561412 CC-CACCCCCCCCC-ACCCCCCCCC-C-CCCC   81 1561429 CC-CTCTCCTCCCCCTCCCCCCCCC--CCCCC   82 1561461 AARAGAAAAAAAAAAGAAAAAA-AAAAAA-AA   83 1561493 AAAAAAAAAAAAAAA-AAAAAAGGAGAAA-GA   84 1561533 CC-C-C--C--CCC---CCCCCAA--CCC-AC   85 1561651 CT-T-CTC-TCTTTT-TTCCCCCCC-CCCCCC   86 1561706 CC-CTCC-CCCCCCCTCC-CCCCCC-CCC-CC   87 1561766 T----T-TT-T-AA-AA-TTTT--TTTTTT-T   88 1561783 AAAA-A-AA-A-AA-A-AAAAAGGARAAAA-A   89 1561792 TTCT-T-TTTT-TTTC-TT-TTTTTTTTTT-T   90 1561828 ACGCAA-AACA-CCCGCCAAAACCAAAAAACA   91 1561849 GGAGAGGGGGGGGGGAGGGGGGGGGGGGGG-G   92 1561866 CC-CACCCCCCCCCCACCCCCCCCC-CCCC-C   93 1561983 GGG-GG-GGGGARGR-R-GGGGGGGGGGGGGG   94 1561989 ATTATA-AAAAWWWATW-AAAAAAAAAAAAAA   95 1562128 GGGGSG-GG-GGGGG-GGGGGGSGGGGGGGGG   96 1562155 AATATATAA-WAAAATAAAAAATTAWAAAATA   97 1562239 CACACC-CC-CAAAACA-CCCCCCCCCCCCCC   98 1562453 CCCCCTCTC-YCCCCCCCTCCCCCCTTCTCCT   99 1562660 GGGGG--CGGCGGG-G--CGGGGGGSCGCGGC  100 1562719 ATTTT-TTA-TTTTTTT-TAAAAAAWTATAAT  101 1562751 GTGTGGGGG-GTTTTGT-GGGGGGGGGGGGGG  102 1562768 GGSGCGGGG-GGGGGCG-GGGGGGGGGGGGGG  103 1562844 AG-GGG-GAGRGGG-GGGGAAAAAARGAGAA-  104 1562877 A-ATAWAWAAAW--AAA-AAAAAAAAWAAAAA  105 1562884 A-A-AAGAAGAAAAAAA-AAAAAAAAAAAAAA  106 1563239 TTTTTKT-TTTTTTTTTTTTTTTTTK-TKTT-  107 1563245 GGGGGTGTGGGGGGGGGGTGGGGGGK-GTGG-  108 1563541 AGGGGG-GA-AGGGGGGGGAAAAAARGAGAAG  109 1563768 AAAAACACAAAAAA-AAACAAAAAAMCACAAC  110 1563924 GTTTTTTTGTKTTTTTTTTGGGGGGKTGTGGT  111 1564092 AAAAAATAATAAAAAAA-AAAAAAAAAAAAAA  112 1564318 GTGTGGGGGGGTTTTGTTGGGGGGGGGGGGGG  113 1564390 CTCTCCCCCCCTTTTCTTCCCCCCCCCCCCCC  114 1564756 AACAAAAAAAAAAAACAAAAAAAAAAAAAAAA  115 1564816 CCMCACCCCCMCCCCACMCCCCCCCCCCCCCC  116 1565451 CCTCTTC-CCYCCCCTCCTCCCCCCYTCTCCT  117 1565457 TTGTGGT-TTKTTTTGTTGTTTTTTKGTGTTG  118 1565592 GGTGTTGTGGTGGGGTGGTGGGGGGKTGTGGT  119 1565646 TTTTTTGTTGTTTTTTTTTTTTTTTTTTTKTT  120 1565826 TTAT-A-AT-WTTTTAT-ATTTTTTWATATTA  121 1565857 CT-T----C-CTT---T--CCCCCCC-C-CC-  122 1565858 TC-C----C-C-C---C--CTCCCCC-C-CC-  123 1565931 A------AA-AG---G---AAAAAA--A-AA-  124 1565944 T--C----T-TCC--T---TTTTTT--T-TT-  125 1566440 C--CG---C-CCCC-CCC-CCCCCC-SCSCCS  126 1566449 G-GTG---G-GTT---TT-GGGGGGGGGKGGG  127 1566453 CCTCTC--CCYCCC-TCCCCCCCCCCCCCCCC  128 1566550 GGCGCCGCGGSGGGGCGGCGGGGGGSCGCGGC  129 1566626 GGKGTGGTGGKGGGGTG-GGGGGGGGGGGGGG  130 1566638 TTKTGTTTTTKTTTTGT-TTTTTTTTTTTTTT  131 1566726 GGTGTTGTGGKGGGGTGGTGGGGGGKTGTGGT  132 1566728 TTCTCCTCTTYTTTTCTTCTTTTTTYCTCT-C  133 1566793 TTCT-CTCTTTTTTTCTTCTTTTTTYCTCTTC  134 1566821 T-CT--TCTTTTTTTCT-CTTTT-T--TCTTC  135 1566823 A-CC--CCACACCCCCC-CAAAA-A--ACAAC  136 1566867 T-CT-CT-TTTTTTTCT--TTTTT---T-TT-  137 1566882 C-CC-CT-CTCCCCCCCC-CCCCC---CCCC-  138 1566890 G-AG-AG-GGGGGGGAGG-GGGGGGG-GAGG-  139 1566891 C-CT-CC-CCCTTTTCTT-CCCCCCC-CCCC-  140 1566906 TTTT-YT-TTTTTTTTTT-TTTTTTTC-YTTC  141 1566911 GG-G-GA-GAGGGGG-GG--GGGGGGG-GGGG  142 1566963 TC-C----T-TCC---C--TTTTTTT-T-TT-  143 1566964 TC-C----T-TCC---C--TTTTTTT-T-TT-  144 1566975 T--T----TCTCCCC-C--TTTTTTT-T-TT-  145 1567049 GGAGGAGGG-GGGGGGGGAGGGGG-AAGAGGA  146 1567111 TATATTTTT-TAAAATAA-TTTTTTTTTT-TT  147 1567133 GGAG-AGAGGRGGGGAGGAGGGGGGRAGA-GA  148 1567183 CCTCTTCTCCYCCCCTC-TCCCCCCTTCTCCT  149 1567261 TTCTCTTCT--TTTTCTTTTTT-TTTTTTTTT  150 1567327 GGAGAAGAGGRG-GGAGGAGGGGGGRAGAGGA  151 1567332 ACCC-CCCACMC-CCCCCCAAAAAAMCACAAC  152 1567385 ACACAAAAAAACCCCACCAAAAAAAAAAAAAA  153 1567427 G-GGGG-GG-GGTKKGGGGGGGGGG-G-GGKG  154 1567428 T-TTTT-KT-TTGKKTT-TTTTTTT-T-TTKT  155 1567438 TTCTC--TT-TTTTTCT--TTTTTT--TCTT-  156 1567439 TTCTC---T-TYTTYCT--TTTTYT--TCTY-  157 1567449 TTGTGGTTTTTTTTTGT--TTTTTT-GTGTTG  158 1567459 CCACAACCCCCCCCCAC--CCCCCC-ACACCA  159 1567536 GAGAGG-GGGGAAAAGAAGGGGGGGGGGGGGG  160 1567581 GGGGGA-GG-GGGGGGGGAGGGGG-GAGAGGA  161 1567585 CCTCTT-CC-YCCCCTCC-CCCCC-CTCTCCT  162 1567588 TTTTTCTTT-TTTTTTTT-TTTTT-TCTCTT-  163 1567602 AGGGGGGGA-RGGGGGGGGAAAAAARGAGAAG  164 1567636 AAGAGAAAAAAAAAAGAAAAAAAAAAAAAAAA  165 1567642 TTTTTATTTTTTTTTTTTATTTTTTWATATTA  166 1567648 AAGAGGAAAAAAAAAGAAGAAAAAARGAGAAG  167 1567659 GGGGGAGGGGGGGGGGGGAGGGGGGRAGAGGA  168 1567661 CCCCCCTCCTCCCCCCCCCCCCCCCCCCC-CC  169 1567665 CC-CCCTCCTCCCCCCCCCCCCCCCCCCC-CC  170 1567673 GG-GAGGGGGRGGGGAGGGGGGGGGGGGGGGG  171 1567679 TTCTCCTTTTYTTTTCTTCTTTTTTTCTCTTC  172 1567685 CCCCCCTCCTCCCCCCCCCCCCCCCCCCCCCC  173 1567691 TTTTTGTTTTTTTTTTTTGTTTTTTTGTGTTG  174 1567714 AAAAAGAAAAAAAAAAAA-ARAAAAAGAGAAG  175 1567716 CCCCCTCCCCCCCCCCCC-CCCCCCCTCTCCT  176 1567728 TTYT-CTTTTTTTTTCTT-TTTTTTTCTCTT-  177 1567768 CCTCTC-CCC-CCCCTCCCCCCCCCCCCCCCC  178 1567770 CCCCCT-CCC-CCCCCCCTCCCCCCYTCTCCT  179 1567788 CCCCCT-CCCCCCCCCC-TCCCCCCYTCTCCT  180 1567986 ATTTTT-TATWTTTTTTTTAAAAAAWTATAAT  181 1568005 AGGGG-AAAARGGGGGGGGAAAAAAR-AGA-G  182 1568012 GGGGG-GGGGGGGGGGGG-GGGGGGG-GAGGA  183 1568019 AAAAA-AAAAWAAAAAAA-AAAAAAA-ATAA-  184 1568021 CTCTC-CCCCCTTTTCTT-CCCCCCC-C-CC-  185 1568085 ATTTT-AAAAWTTTTTTT-AAAAWAA-A-AA-  186 1568120 AAGAG-AAAAAAAAAGAA-AAAAAAA-A-AA-  187 1568124 CCTCT-CCCCSCCCCTCC-CCCCCCC-C-CC-  188 1568168 AAAAA-AAAAAAAAAAAAAAAAAAAM-ACAAC  189 1568196 GGRGAGGGGGGGGG-AGGGGGGGGGGGGGGGG  190 1568214 AARAAGAAAARAAAAAAAGAAAAAARGAGAAG  191 1568478 GGGGGCGGGGSGGGGGGGCGGGGGGSCGCGGC  192 1568490 AAAAAACAACAAAAAAAAAAAAAAAAAAAAAA  193 1568548 TTWTTATTTTTTTTTTT-ATTTTTTWATATTA  194 1568634 TTTTTATTTTTTTTTTTTATTTTTTTA-ATTA  195 1568727 TTTTTGTTTTTTTTTTTTGTTTTTTTGTGTTG  196 1568784 CCCCCG-CCCCCCCCCC-GCCCCCCCG--CC-  197 1568820 GGGGGA-GGGGGGGGGG-AGGGGGGGAGAGG-  198 1568826 AAAAAG-AAAAAAAAAA-GAAAAAAAGAGAA-  199 1568868 CCCCCACCCC-CCCCCC-ACCCCCCCA-A-C-  200 1568870 TTTTTGTTTT-TTTTTT-GTTTTTTTG-G-T-  201 1568916 G--G-G--G-GG---GG--GGGAA-G-GGGA-  202 1568919 G--K-T--G-GG---GG--GGGGG-G-G-GG-  203 1568929 A-GAG---AGAAA-AGA--AAAAA-A-A-AA-  204 1568939 G-GGG---GGGGG-GGG--GGGGG-G-G-GGA  205 1568952 C-CTCC--CCCTT-TCT-CCCCCC-CCC-CCC  206 1568963 G-GGGA--GARGGGG-G-AGGGGG-GAG-GGA  207 1569035 GG-G-TTGGTKGGGG-GG-GGGGGG--G-GG-  208 1569058 CTCTCCCCC-CTTTTCT--CCYCCC-CCCCCC  209 1569074 TTTTTCTTTTTTTTTTTTCTTTTT--CTCTTC  210 1569093 AAAAACAAAAAAAAAAAACAAAAA-ACACAAC  211 1569128 CCCCCTCCCCCCCCCCCC-CCCCC-CTCTCC-  212 1569136 TTTTTCTTTTTTTTTTTT-TTTTTTTCTCTT-  213 1569138 TTCTCTTTTTTTTTTCTT-TTTTTTTTTTTT-  214 1569140 AAAWATAAAAAAAAAAAA-AAAAAAATATAA-  215 1569146 CCCCCCTCCTCCCCCCCC-CCCCCCCCCCCC-  216 1569167 AGGGG-AGAARGGGGG-G-AAAAAAA-AGAA-  217 1569185 GGAGA-GGGGRGGGGA-G-GGGGGGG---GG-  218 1569190 CTCT--TCC-CTTTTC-T-CCCCCCC---CC-  219 1569227 CAA-----C-CA--A----CC-CCCC---CC-  220 1569374 TCCCCCC-T--CCC-CCCCTT-TTT--T-TT-  221 1569395 GGGGGAG-GGGGGG-GGGAGGGGGG--GAGG-  222 1569396 GGAGAGG-GGGGGG-AGGGGGGGGG--GGGG-  223 1569405 GGGGGTG-GGGGGG-GGGTGGGGGGT-GTGGT  224 1569441 TTTTTTTTTTTTTTTTTTTTTTTTT-GTGTT-  225 1569442 GGGGGKGGGGGGGGGGGGGGGGGGG-TGTGG-  226 1569557 TTWTATTTT--TTTTTT-TTTTTTTWTT-TTA  227 1569564 AAWAA-AAA-AAAAAWA-TAAAAAAAWA-AAA  228 1569704 TTWTATTTTTTTTTTAT-TTTTTTTTTTTTTT  229 1569788 C-CCCGCCCCCCCCCCCCGCCCCCC-GCGCCG  230 1569791 G-GGGTGGGGGGKKGGGG-GGGGGG--GTGGT  231 1569794 T-TTTWTTTTTTTTTTTTATTTTTT-ATTTTT  232 1569797 A-AAATAAAAAWWWAAWAGAAAAAA-AATAAT  233 1570104 AAAA--AA--AAAA-AA--AAAAAA--A-AAT  234 1570126 GGGG-CGGG-GGGG-GG-CGGGGGGSCGCGGC  235 1570416 GGTGTGGGGGGGGGGTGGGGGGGGGGGGGGGG  236 1570660 TTTTTTCTTCTTTTTTTT-TTTTTTTTTTTTT  237 1570881 TTTWTT-T-TTWWWATWW--TTTTTTTTTTTT  238 1571274 TTYTTC-TTTTTTT-TTTCTTT-TTYCTCTTC  239 1571487 TCCCCCCCTCYCCCCCC-CTTTTTTYCTCTTC  240 1571774 AGGGGGGGA-GGGGGGGGGAAAAAARGAGAAG  241 1572279 GAAAAAAAGARAAAAA-AAGGGGGGRAGAGGA  242 1572432 TT-TTC-TTTTTTT-TT--TTTTTTYCTCT-C  243 1572888 T--TGTTTT-TTTTTGT-TTTTT-TTTTTTT-  244 1572987 ACCCCCCC-CMCCCCCCCCAAAAAAMCAC-AC  245 1573060 CGGGGGGGCGGGGGGGGGGCCCCCCSGCGCCG  246 1573221 ACCCCCCCACMCCCCCC-CAAAAAAACACAAC  247 1573239 AAMACA-AAAAAAAACA-AAAAAAAAAAAAAA  248 1573328 GGCGGC-GGGGGGGGGGGCGGGGGGCCGCGGC  249 1573482 CCTCTC-CCCCCCCCTCCCCCCCCCCCCCCCC  250 1574247 AATAATAAAAAAAAAAAATAAAAAAWTATAAT  251 1574545 GGGGGTGGGGGGGGGGGGTGGGGGGKTGTGGT  252 1575684 TCTCTT-TTTTCCCCTCCTTTTTTTTYTTTTT  253 1575961 CGCGCCCCCCCGGGGCG--CCC-CCCCCCCCC  254 1576052 G--G-A--GGGAAG-A---GGGGGGGGG-GG-  255 1576055 TG-K-G--TTTGGT-G---TTTTTTT-T-TT-  256 1576059 CCCS-C--C-CCCG-C---CCCCCCCCCCCC-  257 1576291 CCCC-TC-CC-CCCCC-C--CCCCCCTCTCC-  258 1576327 A----A--A----T-----WWAAAA----AA-  259 1576345 A-------A-A-TAT----AAAAAA----AA-  260 1576372 AACACA--A-A-AAAC-A-AAAAAAAAAAAAA  261 1576552 A-T-TT-AAAW-TTTTTTTAAAAAATT---AT  262 1576569 G-G-GG--GGG-GRGG-AGGGGGGGG----G-  263 1576597 GGGGGG--GGGGGGGGGGAGGGGGG-A-A-R-  264 1576682 GTTTTTTTGTGTTTTTTTTGG-GGGGT-TGGT  265 1576719 GGRGGAG-GGGGGGGGGGRGGGGGGGAGAGGA  266 1576755 G-A--G--G-G-AAAA---GGGGGGGGGGGGG  267 1576816 ACACAAA-AAACCCCACCAAA-AAAAAAAAAA  268 1576824 CTCTCCCCCCCTTTTCTTC-C-CCCCCCCCCC  269 1576881 CC-CCTCCCCCCCCCCCCT-CCCCCTTYTCCT  270 1577186 AAA-AATAATAAAAAAA-AAAAAAAAAAAAA-  271 1577187 AAA-AATAAT-AAAAAA-AAAAAAAAAAAAA-  272 1577188 AAA-AATAATTAAAAAA-AAAAAAAAAAAAA-  273 1577205 ACC-CCCCACACCCCC--CAAAAAAMCACAAC  274 1577558 T----A--T-TT-T-TTT-TTTTTTTAT-TTT  275 1577559 T----A--T-TT-T-TTT-TTTTTTTAT-TTA  276 1577560 A----A--A-WT-T-TTT-AAAAAAAAA-AAA  277 1577562 A----T--A-AA-A--AA-AAAAAAA-A-AAT  278 1577563 A----T--A-AA-A--AA-AAAAAAA-A-AAT  279 1577633 TT-T-TG-TGTTTTTT-T-TTTTTTTTTTTTT  280 1577638 AG-G-AA-AAAGGGG--G-AAAAAAAAAAAAA  281 1577661 TT-T--C-T-TTTTT-TT-TTTTTTYC-CTTC  282 1577669 TT-T--G-T-TTTTT-TT-TTTTTTTGT-TTG  283 1577673 CT-T--C-CCCTTTT-TT-CCCCCCCCC-CCC  284 1577684 TC----C-TCYCCCC-CC-TTTTTTT-T-TT-  285 1577691 TC-C----TCTCCCC--C-TTTCTTT-T-TT-  286 1577708 GG-GC---GCGGG-G--G-GGGGGGGCG-GG-  287 1577712 TC-CCTT-TTTCC-C--C-TTTTTTTTT-TT-  288 1577745 CCCCCAC-CCCCCCCC-CACCCCCCCA-ACCA  289 1577746 GGRGGAG-GGGGGGGGGGAGGGGGGGA-AGGA  290 1577755 GCGCGGG-GGGCCCCGCCGGGGGGGGG-GGGG  291 1577762 GAGAGGG-GGGAAAAGAAGGGGGGGGG-GGGG  292 1577765 AAWATAA-AAAAAAATAAAAAAAAAAA-AAAA  293 1577792 GGTGTG--GGAGGGGTGGGGGGGGGGGGGGGG  294 1577795 CGCGCC--CCCGGGGCGGCCCCCCCCCCCCCC  295 1577857 TWTTTTTTTTTTATTTWTTTTTTTT-TTTTTT  296 1577867 CCCTCCCCCCCC-TTCTTCCCCCCCCCCCCCC  297 1577890 TTTTTTTGTTKTTTTTTTTTTTTTTTTTTTTT  298 1578154 TCCCCC-CTCYCCCCCCCCTTTTTTYCTCTTC  299 1578364 TATATT-TT-TAAAA-AA-TTTTTTT-T-TTT  300 1578462 AAAAAG-AAAAAAAAAAAGAAAAAARGAGAAG  301 1578538 TTYTTC-TTTTTTTTTTTCTTTTTTYCTCTTC  302 1578727 GGGGGGTGGTGGGGGGGGGGGGGGGGGGGGKG  303 1578925 AAAAAAACAAMAAAAAAAAAAAAAAAAAAAAA  304 1579270 AAAAAAGAAGAAAAAAAAAAAAAA-AAAAAAA  305 1579346 TGKG-TGGTGKGGG-GGGT-TTTTTTTT-TTT  306 1579707 TTTTTTGTTGTTTTTTTTTTTTTTTTTTTTTT  307 1579708 CCCCCCTCCTCCCCCCCCCCCCCCCCCCCCCC  308 1580305 CTTTTTTTCTYTTT-TTTTCCCCCCYYCTCCT  309 1581345 GGRGAGGGGGGGGGGAGG-GGGGGGGGGGGGG  310 1581602 TCCCCCCCTCYCCCCCCCCTTTTTTCCTCTTC  311 1581762 TTYTTCTCTTTTTTTTTTCTTTTTTTCTCTTC  312 1581931 TTTTTGTTKTTTTTTTTTGTTTTTTKGTGTTG  313 1582195 CTTTTTTTCTYTTTTTTTTCCCTTCYTCTCTT  314 1582351 AGGGGG-GAAAGGGGGGGGAAAAAAR-AGAAG  315 1582357 GAAAAA-AGGGAAAAAAAAGGGGGGRAGAGGA  316 1582363 CTT-TTTTCCCTTTTTTTTCCCCCCYTCTCCT  317 1582479 A-GA-A-AAAAAA-AG--AAAAAAAAAAAAAA  318 1582483 A-AA-R--AAA---AA--AAAAAAAARAGAAA  319 1582484 T-TT-W--TTT---TTA-TTTTTTTTWTATTT  320 1582487 T-GT-K--TTT-T-G-T-GTTTTTTTTTTTTG  321 1582566 AAAAAAAAATTAAAAAAAAAAAAAAAAAAAAA  322 1582570 TCCCCCCCTTTCCCCCCCCTTTTTTYCTCTTC  323 1582623 G--G-K--G-G-G---KT-GGGGGGK-G-G--  324 1582625 G--G-K--G-G-G---KTGGGGGGGG-G-G--  325 1582636 TT-T-A--T-T-TT-TTTATTT---W-T-T--  326 1582637 TT-T-G--TTT-TT-TTTGTTT---K-T-T--  327 1582694 CC-C-CCCCCCCCC-CCCCCCC-ACCCC-CAC  328 1582722 GAGG-G--GG-------AAGGG-RGGAG-GG-  329 1582723 ATAA-A--AA-----A--TAAA-WAATA-AA-  330 1582737 G-GG-G-GGG-----G--GGGG-AGG-G-GA-  331 1582739 A-GA-G-GAA-A---A--GAAA-AAA-A-AA-  332 1582767 C-TT-T-TCCCT---T--TCCC-CCC-C-CCT  333 1582785 A-G-----AAA--------AAA-AAA-A-AAG  334 1582786 T-G-----TTT--------TTT-TTT-T-TTG  335 1582824 GGGGGGG-GGGGGG----GGGG-AGGGG-GAG  336 1582825 CCCCCCC-CCCCCC-C--CCCC-TCCCC-CTC  337 1582843 AGGGGGGGAAAGGG-GGGGAAAAAARGAGAAG  338 1582860 GAAAAAAAGGGAAA-AAAAGGGGGGGAGAGGA  339 1582871 CCCCCCCCCCCCCCCCCCCCCCTTC-CCCCTC  340 1582941 AAAAAAAAAAAAAAAAAAAAAACCAAAAAACA  341 1582946 TAAAAATATAWAAAAAAAATTTAATWATATAA  342 1583115 AAAAAAAAAAAAAAAAAAAAAARGAAAAAAGA  343 1583431 GCCCCCCCGCSCCCCCCCCGGGCCGSCGCGCC  344 1583461 GAGAGG-GGGGAARAGAAGGGGGGGGGGGGGG  345 1583655 TTTTTTG-TTTTTTTTTT-TTT--TK-T-T--  346 1583764 CCMCCAAACCCCCCCCM-ACCCAACMACACAA  347 1583859 GGGGGTTTGGGGGGGGG-TGGGTTGKTGTG--  348 1583939 ATWTAT-TAAA-TT-AT--AAATTA-TATATT  349 1584144 TAWATAAATAWAAAATA-ATTTAATWATW-AA  350 1584266 TKTTTT-KT-T-TT-T--TTTT-TTTTT-TTT  351 1584267 GRGGGG-RG-G-GG-G--GGGG-RGGGR-G-G  352 1584541 AAGAGG-GAGAAAAAGAAGAAAGGARGAGAGG  353 1584669 AGGG-GGGAGGGGGGGR-GAAAGGARGAGAGG  354 1585055 TTTTTA-ATTTTTTTTTTATTTTTTWWTATTA  355 1585295 TTTTTT-AT-TTT---TT-TTTTTTT-T-TA-  356 1585304 T-TW---TTTT-WT--T-TTTT--TT-T--T-  357 1585332 T-GG---GTGT-GG-GG-GTTT--TK-TG-G-  358 1585543 GAAAAAAAGAAAAAA-AAAGGGAAGRAGAGAA  359 1585768 TA--AA-A-AT-AA-AT-AT--AA---TAT--  360 1586016 TA--TT-TT-T-WT-TT-TTTTTTTT-TTTT-  361 1586018 AT--AA-AA-A-WA-AA-AAAAAAAA-A-AA-  362 1586074 AA-AT--WA-AAAA-AA-AAAAAAAA-A-A--  363 1586080 AM-MAA-AA-MCAC-CM--AAA--AA-A-A--  364 1586082 CM-MCC-CC-C-CA-AM--CCC--CCCC-C--  365 1586217 ATTTTT-TATWTTTTTT--AAATTAWTATATT  366 1586324 TAT-TAATTT---AAT-A-TTTTTTW-TATTA  367 1586325 AAA-AAAAAA---AAA-A-AAAAAAW-AWAAT  368 1586334 ACC-MCCCACC--CCC-C-AAACCAACACACC  369 1586942 GTTT-TT-GTTTTTTTTT-GGGTTGGTGTGT-  370 1586943 TAAA-AA-TAAAA-AAAA-TTTAATTATATA-  371 1586945 TGGG-GG-TGGGGGGGGG-TTTGGTTGT-TG-  372 1587141 GTTTTT-TGTKTTTTTTTTGGGTTGKTGTGTT  373 1587173 CTTTTT-TCTCTTTTTTTTCCCTTCYTCTCTT  374 1587518 CTTTTTTTCTYTTTTTTTTCYCTTCYTCTCTT  375 1587643 GTKTTTTTGTGTTTTTTTTGGGTTGKTGTGTT  376 1588896 AAWATAAA--AAAAATAAAAAAAAAAAAAAAA  377 1589020 ATATATTAAAATTTTATTTAAAAAAWTATAAT  378 1589177 T-T-T--WTT-TTW--W-TTTTA-TT-TWTTA  379 1589187 G--GAG-GGR-R-G--G-GGGGGGGG-GGG-G  380 1589259 TTATATTTTTTTTTTAT-TTTTTTTTTTT-TT  381 1589715 GTTTTTTTGTKTTTTTTTTGGGTTGKTGTGTT  382 1589780 AT-TTTTTATTTTT-TT-TAAATTAWTATATT  383 1589870 TTTTTTTTTTTTTTTTTTTTTTCCTTTTTTCT  384 1589938 CCGSGCCCCCCCCCCGC-CCCCCCCCCCCCCC  385 1591968 GTGKGGGGGGGGGGGGGGGGGGGGGGGGGGGG  386 1592485 GGAGAGG-GGGGGGGAGG-GGGGGGGGGGG-G  387 1592711 TYYTTTTTTTTTTTTTTTYTYTTTTT--TTTT  388 1592832 TYCTTTYTYTTTTT-YYTTTYTYCTTYTTYTY  389 1592838 CYYCCCYCYCCCCC-YCCCCYCYTCCCCCCCY  390 1593700 AAAAAAAAAAAAAA-AAAAAAACCAAAAAACA  391 1593863 AAWATAAAA-AAAA-TAAAAAATTAAAAAATA  392 1594079 TTWTATTTTTTTTTTATTTTTTT-TTTTTT-T  393 1594162 GGKGTGGGGGGGGGGTGGGGGGGGGGGGGGGG  394 1594233 TTYTCTTTTTTYTTTCTTTTTTTTTTTTTTTT  395 1594426 A-GGGGG-AGRGGGGGGGGAAAGG-RGAGAGG  396 1594480 T-TYTCTTTTTCCCCTC-CTTTTTTYCTCTTC  397 1594800 A-TATAAAAAAAAAATAAA-AAAAAAAAAAAA  398 1594961 GGGKGTGG-GGTTTTGTTTGGGGGGKTGTGGT  399 1594983 AAWATAAAAAAAAAATAAAAAAAAAAAAAAAA  400 1595159 TTTTTTTTKTKTTTTTTTTTTTTTKTTTTTTT  401 1595360 AAARAGA-AAAGGGGAGGGAAAAAAGGAGAAG  402 1595545 GGGGG-GGGGG-AR-G-A-GGGGGG--G-GG-  403 1595560 TTCTCTTCTT-TTT-CTT-TTTTTT-TT-TT-  404 1595571 CCTCTCCTCC-CYC-TCC-CCCCCC-CCCCC-  405 1595887 CT-Y-C--CTCCCC-C-C-CCCCCC-CCCCCC  406 1595916 TTCTCT-CTTYTTTT--T-TTTTTTTTTTTTT  407 1595942 AAGAGAAGAAAAAAAGAAAAAAAAAA-AAAA-  408 1596204 TT-TCTT-TYTTTTTCTTTTTT-TTTTTTT-T  409 1596317 TTCTCTT-TTY-TTTCTTTTTTTTTTTT-TTT  410 1596434 AATATA--AWTAAAATAAAAAAAAA-AAAA-A  411 1596445 AATATA--AWWAAAATAAAAAAAAA-A-AA-A  412 1597089 CAAAAAAAM-AAAA-AAAACCCCAC-ACACA-  413 1597188 TAAAAAATTATAAAAAAAATTTTATTATATAA  414 1597206 ATWT-TTAA--TTTTTTTTAAAATAWTATATT  415 1597307 CTTTTT-CC-CTTT-TTTTCCCCTCTTCTCTT  416 1597320 TCCCCC-TT-TCCC-CCCCTTTTCTCCTCTCC  417 1597401 CCCCCTCCCCCTTTTCTTTCCCCCCY-CTCCT  418 1597531 G-GC-C-GGCG---C-C--GG-G-GG-G-G-C  419 1597534 G-GA-G-GG-G----GA--GG-G-GG-GGG-A  420 1597566 A-TTTT-AATATTT-TTTTAA-ATAWTATATT  421 1597599 TAWAAAATTATAAAAAAAATTTTATWATATAA  422 1597812 CCYCTCCCCCCCCCCTCCCCCCCCCCCCCCC-  423 1597849 TCCCCC-TTCTCCCCCC-CTTTTCTYCTCTC-  424 1597865 ATTTTT-AATATTT--T-TAAAA-AATA-AT-  425 1597868 A-TAAA-A-AA-AA--W-AAAAA-AA-ATAA-  426 1597869 G-AGRG-G-GG-GG--R-GGGGGAGG-GAGR-  427 1598084 AAAWA-AAAAA----A---AA-AAAA-ATAAT  428 1598085 CCCMC-CCCCC----C---CC-CCCC-CACCA  429 1598141 GCGGGGGGG-G-GG-GGGGGGGGCGGGGGG-G  430 1598160 G-AA-A-GG-G--A-AAAAGGGG-GGAGAG-A  431 1598175 GGTT---TG-K-GT--TT-GGG--G--GTG--  432 1598279 ACMCCCAAACACCCCCCCCAAAACAMCACACC  433 1598409 A-A--AAAA-A-AATAA--AAAAAAA---AT-  434 1598416 TGTG-KTTKGT--TGTT--TTTTTTTG--TGG  435 1598417 AAAA-AAAAAA--TAT---AAAA-AAA-AAAA  436 1598418 TTTT-TTTTTT--ATW-T-TTTTTTTT-TTTT  437 1598562 TGGGGGTTT-TGGGGGGG-TT-TGTTGTGTGG  438 1599197 T-W-TT---WTT-T-----T--TT----TT-T  439 1599227 CGCGCCC--CCCCCCCC-CCCCCC-C-CCC-C  440 1599306 TCCCCC-T-CTCCCCCCCCTTTTCTYCTCTCC  441 1599529 A---T-AAA-A--------AAAA-AA-A-A-T  442 1599531 GA--A-GGG-G-A------GGGGAGG-G-G-A  443 1599532 ACC-C-AAA-ACC------AAAACAA-A-A-C  444 1599608 CTYTTTCCCTCTTTTTTTTCCCCTCYTCTCTT  445 1599686 TGKGGG-TTGTGGGGG-GGTTTTGTK-TGTGG  446 1599688 TCYCCC-TTCTCCCCCCCCTTTTCTY-TCTCC  447 1599708 GGGGAG-GGGGGG--AGGGGGGGGGG-GGGGG  448 1599712 GG-GGG-GGGGGG--GGGGGGGGAGG-GGGAG  449 1599720 CG-GGG-CCGCGGG-GGGGCCCCGCS-CGCGG  450 1599826 T-A-AGTTT-T--GGA--GTTTTGTT-T-TGG  451 1599827 C-C-CTCCC-C--TTC--TCCCCCCC-C-CCT  452 1599828 G-R-AAGGG-G--A-A--AGGGGAGG-G-GA-  453 1599836 T-K-GGTTT-T----G--GTTTTGTK-T-TG-  454 1599868 T---T--CT-Y-C------TTTTCTY-TCT--  455 1599869 G------GG-G-A------GGGGAGR-GAG--  456 1599900 G------GG-G--------GGGG-GG-GCGC-  457 1599907 AG-G---GA-RG-G-GG-GAAAA-AA-AGAG-  458 1599975 A-AG-GAAA-AGG-GGG-GAAAAGAAGAG-GG  459 1599986 C-C---CCC-C-T-TCT-TCCCC-CCTCTCCT  460 1599993 G-G---GGG-G----AA--GGGG-GG-G-G--  461 1600015 G-----GGG-G--A-A---GGGG-G--GAGA-  462 1600017 G-----GGG-G--A-A---GGGG-G--GAGA-  463 1600060 CGG-G-ACC---GG-----CCCC-CSGCGC--  464 1600072 G-C---GGG-G-CC-----GGGG-GGCG-G--  465 1600084 T----CTTT-T-CC--C--TTTT-TT-T-T--  466 1600128 C-C-TCCCC-CC-CCTCC-CCCC-CC-CCCC-  467 1600162 C-C---CCC-C-G-G----CCCC-CC-C-C--  468 1600179 A--TT-TAA-ATTTTTTT-AAAA-AW-A-A-T  469 1600193 T--CCCCTT-TCCCCCCC-TTTT-TY-T-T-C  470 1600209 G-GGGGGGG-GGGGGGGGGGGGG-GGCG-G-C  471 1600945 TCYCCCCTTCTCCCCC--CTTTTCTCCT-TCC  472 1600951 CTYTTTTCCTCTTTTT--TCCCCTC-TCTCTT  473 1600980 CTYTTT-CCTCTTTT-TTTCCCCTC-TCTCTT  474 1600987 TTWTATTTTTTTTTTATTTTTTTTT-TTTTTT  475 1601238 AATATAAAAAAWAAATAAAAA-AAAAAAA-AA  476 1601551 ATTTTTTTA-WTTTTTTTTAAAATAWTATTTT  477 1602219 GGGKGTGGGGGTTTTGTTTGGGGGGKTGTGGT  478 1602244 CCSCGCGGCCCCCCCGCCCCCCCCCCCCCCCC  479 1602297 ATATAAAAATAAAAAAAAAAAAATAAAAAATA  480 1602308 ACAMAAAAACAAAAAAAAAAAAACAAAAAACA  481 1602593 GGGGGGGGG-GTGG-K---GGG--G--G-G-G  482 1602594 ATAWTTAAA-AA-A-T---AAA--A--A-A-T  483 1603109 TTTT-ATTTTT-A-T-T--TTTT-TWAT-TA-  484 1603138 AA-A-TAAAAA-----A--AAAA-AAA--AA-  485 1603142 AG-R-GAA-AAGGG-----AAAAGAAAA-AA-  486 1603143 AA-A-AAA--AAAA-----AAAAAAW-A-AT-  487 1603220 CCCYCTC-CCCTTTT-TTTCCCCTCYTCTC-T  488 1603235 CCCYCTC-CCCTTTTCTTTCCCCTCYTCTC-T  489 1603332 TTYTCTTTTTTTTTTCTTTTTTTTT-TTTTTT  490 1603367 CTTTTTCCCTCTTTTTTT-CCCCTC-TCTCTT  491 1603440 TTTT----TTTTKT-T-G-TTTT-TTTT-TK-  492 1603441 GGGG----GGGGGG-G-A-GGGG-GGGG-GR-  493 1603653 AAAAAGAAAAAGGGGAG-GAAAAGARGAGAGG  494 1603713 AGG-GGGGA-GGGGGGGGGAAA-GARGAGAGG  495 1603719 ATA-AAAAA-TAAAAA-AAAAAAAAAAAAAAA  496 1603723 CCC-CGCCC-CGGGGC-GGCCCCGCSGCGCGG  497 1603741 TTTTT-TTT-T--CCT---TTTTCTYCT-TCC  498 1603750 TTTTT-TTT-T----T---TTTT-TYCT-TCC  499 1603771 GGGGG-GGG-G--C-G---GGGG-GG-GC--C  500 1603774 CCTCTCCCC-C--C-T---CCCC-CC-CC--C  501 1603778 TCCCCCCCT-Y--C-C---TTTT-TT-TC--C  502 1603794 GGGGGAGGGGGAAA-GA--GGGGAGGAGA-AA  503 1603797 TCCCCTCCTCYTTT-CT--TTTTTTTTTT-TT  504 1603800 AAAAAGAAAAAGGG-AG--AAAAGAAGAG-GG  505 1603857 CGARGA-GCGCAAAAGA-ACCCCACCAC-CAA  506 1603877 CTCTCCCCCTY--YYC--YCCCCCCCCC-CCC  507 1603887 AAAAAAAWAAA--AA---A-AAAAAATAAAWW  508 1603952 GGGRGAGGGGGAAAAGA-AGG-GAGRA-AGAA  509 1604000 CCCYCT-CCCCTTTTCTTTCCCCTCYTCT-TT  510 1604145 TATATT-TTAT-TTTT--TTTTTTTTTTTTTT  511 1604181 TTYTCT-TTTT-TTTCTTTTTTTTTTTTTTTT  512 1604183 GGGGGA-GGGG-AAAGAAAGGGGAGRAGAGAA  513 1604206 CCCCCT-CCCCTTTTCTTTCCCCTCYTCTCTT  514 1604236 CCCYCTCCCCCTTTTCTTTCCCCTCYTCT-TT  515 1604259 GGGGGAGGGGGAAAAGAAAGGGGAGRAGAGAA  516 1604304 GGAGAGGG--RGGG-AGGGGGGGGGGGGGGGG  517 1604307 GGGGGAGGG-GAAA-GAAAGGGGAGRAGAGAA  518 1604385 TTTTT--TTTTTTA-TT-ATTTTTTTTTATT-  519 1604387 CCCCC--CCCCCCA-CC-ACCCCCCCCCACC-  520 1604388 AAAAA--AAAATTT-AT-TAAAA-AATATAT-  521 1604389 TTTTT--TTTTCTT-T--TTTTT-TTCTTTC-  522 1604437 CCTCTCCCCCCCCC-TC-CCCCCCCCCCCCCC  523 1604478 CCCCCTCCCCCTTTTCT-TCCCCTCYTCTCTT  524 1604482 TTTTTATTTTTAAAATA-WTTTTATWATATAA  525 1604540 TCTT-T-YT-T--T-T---TTTT-T--TTT--  526 1604541 TTGGT--KT-T----T---TTTT-T--TGT--  527 1604542 GGCCG--SG-G----G---GGGG-G--GCG--  528 1604543 CCA-C--MC-C----C---CCCC-C--C-C--  529 1604568 TTATA-TTT-TT-T-A---TTTTTTTTT-T--  530 1604611 CGGGGGGGCGCGGG-GG-GCCCCGCSGC-CGG  531 1604637 T--TTKTTGT-TGT----TTTTTTTTKT-TT-  532 1604638 A--AAAAAAA-ATW----AAAAA-AAWA-AA-  533 1604653 CCCYCTCCC-CTTT-CT-YCCCC-CYTC-C--  534 1604820 T-T-T-T-TTTT---TA--TTTTATT-T-TAA  535 1604867 AAAMACAAAAACCCCAMCCAAAACAMCACACC  536 1605056 T-TY-CTTTTTCCCCT-CCTTTTCTCCTCTC-  537 1605193 TGTKTTTTT-TTTT-TTTTTTTTT-T-TTTTT  538 1605226 CCCCCACCCCCAAM-CAAACCCCA-CACACAA  539 1605297 GGGGGA-GG-GGGGGGGG-GGGGGGRA--GG-  540 1605327 TTTT-T-TT--GTK--GT-TTTTTTTT-GTTG  541 1605336 TTT-TA-TT--T-T--TT-TTTT-TAT-TTTT  542 1605337 TTT-TC--T--T-T--TT-TTTT-TCT-TT-T  543 1605389 CCCCC--CC-CAAA-CAAACCCCACMACA-AA  544 1605417 GGGG---GG-GAAA-GAAAGGGG-GR-GA-A-  545 1605443 A-AA-A-TAAA----T---AAAA-AA-AAA-A  546 1605445 GA-AAA-AGAG----A---GGGGAGR-GAG-A  547 1605467 GGGGGA-GGGGAAA-GAAAGGGGAGRAGAGAA  548 1605520 GGRGAGGGGGGGGGGAGGGGGGGGGGGGGGGG  549 1605526 AGGGGGAAAGGGGGGGG-GAAAAGARGAGAGG  550 1605527 CCCCCTCCCCCTTTTCT-TCCCCTCYTCTCTT  551 1605559 CGGGGT-GCGSTT---T-TCCCCTCYTCTCTT  552 1605573 GGGGGAGGGGGAAA----AGGGGGGRAGA--A  553 1605598 GGGGGG-GGGGG-G----GGGGG-GG-GGG-A  554 1605606 AAAAAG-AAAAG------GAAAA-AA-A-AGG  555 1605613 GAAAGG-GGAAG------GGGGG-GG-G-GGG  556 1605623 GGAGGG-GGGAG------GGGGG-GG-GGGGG  557 1605629 GCGC-G-GGCGGG--G--GGGGG-GG-GGGGG  558 1605631 CCCC-T-CCCCTT--C--TCCCC-CC-CTCCT  559 1605665 AAAAA-AAAAA-G--A---AAAA-AA-A-AGG  560 1605667 C-CCC-CCCCC-T--C---CCCC-CC-C-CTT  561 1605687 GGGGGAGGGGG--G-G---GGGGGGG-GAGG-  562 1605702 AAAAAAAAAAAATAAAT-AAA-AAAA-AAAAA  563 1605716 GGGGGAGGGGGAAAAGA-AGGGGAGR-GAGAA  564 1605853 CCCYCT-CCCCTTTTCTT-CCCCTCYTYTCTT  565 1605879 CTCTCC-CCTYCCCCCCCCCCCCCCCCC-CCC  566 1605938 TCTCTCT-TCC-CCCTC-CTTTTC-Y-T-T-C  567 1605946 AAAAAGAAAAA-GGGA--GAAAAG-A-A-A-G  568 1605957 C-CCC-CCCCC--T-C--TCCCCTCC-C-C--  569 1605958 C-CCC-CCCCC--T-C--TCCCCTCC-C-C--  570 1605965 A-AAACAAAAA----A--CAAAACAACA-A--  571 1605983 A-AAAGAAAAA-G--A---AAAARAAGRG-G-  572 1605993 G-GGGAGGGGG-A--GA--GGGGRGGARA-AA  573 1606046 TTT-------T----T----TTT-TT-WAT--  574 1606053 GAA-----A-A---------GGG-GG-G-G--  575 1606065 AAA-A---A-R--G------AAA-AA-A-A--  576 1606094 CTY-CT-TC-Y-TTTCT-TCCCC-CCTCT-TT  577 1606158 GCCCCCCCG-SCCCSCCCCGGGGCGSCGCGCC  578 1606358 AGARAAAAA-AAAAAAAAAAAAAAAAAAAAA-  579 1606360 CCYYCTCCC-CTTTTCTTTCCCCTCYTCTCT-  580 1606554 GGGGG-GG-GG-AA-G-A-GGGGAGG-GAG-A  581 1606615 TTTYTCTTTTTCCCCTC-CTTTTCTCCTC-CC  582 1606726 CTTTT-TTCTY-T-C---ACCCCACC-C-C--  583 1606727 AAAAA-AAAAA-A-----GAAAAGAAGA-AG-  584 1606728 GGGGG-GGGGG-G-----TGGGGTGGTG-GT-  585 1606768 CA-AC--CCAM----C---CCCC-CC-C-C--  586 1606777 GGGGG--GGGG-TT-G--TGGGG-GKTGTG-T  587 1607231 TAWWTTTTTAATTTTTTTTTTTTTTTTTTTTT  588 1607235 GCSSGGGGGCCGGGGGGGGGGGGGGGGGGGGG  589 1607243 GTGKGGGGGTTGGGGGGGGGGGGGGGGGGGGG  590 1607470 ATAWAAATATAAAAAAAAAAAAAAAAAAAAAA  591 1607624 AARRAGGAAARGGGGAGGGAAAAGARGAGAGG  592 1607724 CCCCCCCCCCCCCCCCCCCCCCCACCCCCCAC  593 1607885 CCYYCTTCCCYTTTTCTTTCCCCTCTTCTCTT  594 1608109 TTGTGTTTTTTTTTTGTTTTTTTTTTTTT-TT  595 1608326 AAAAAAAGAARAAAAAAAAAAAAGAAAAAAGA  596 1608370 GGGKGTGGGGGTTTTGTTTGGGGGGKTGTGGT  597 1608498 AAAAAACCAAMAAAAAAAAAAAACAAAAAACA  598 1608523 AAAAAAAMAAMAAAAAA-AAAAA-AAAAAA-A  599 1608720 TTTTTTTTTATTTTTTTTTTTTTATTTTTTAT  600 1608832 AGAGAGAAAAAGGGGA--GAAAAAARGAGAAG  601 1609704 TGGG-GGGTGKGGGGGGGGTTTTTTGGTGTTG  602 1609752 GGGGGCGGGGGCGGGGGGCGGGGGGSCGCGGC  603 1610363 T----K------T---G--TT--G----TG--  604 1610368 CC-C-M---C--A---C--A---C-----C-C  605 1610369 AA-A-A---A--C---A--C---A-----A-A  606 1610748 GGGGGG-GGGGGAAAGAAGGGGGGGGGGGG-G  607 1610778 GAGAGGGGGGG-GGGGGGGGG-GGGGGGGG-G  608 1610902 TCCCTTTTTTYTCC-CC-T-TTTCTTTT--TT  609 1611052 A--G--A-A---G-AA-G-AAAAGA----A-A  610 1611054 G--A--A-A---A-AA-A-GGGGAG----G-A  611 1611234 GGKGKGGGGTGKGKKKGG--GGGG-GGGGGGT  612 1611303 AAA--GG-AAAGAAAAAAGAAAAAAAG-GAAG  613 1611422 GAAAGAA-GARAAAAAAAAGGGGAGRAGAGG-  614 1611490 TT-TTTTTTTTTAAAA-ATTTTTATT--TTT-  615 1611491 TT-TTTTTTTTTAAAA-ATTT-TATT--TTT-  616 1611492 TT-TTTTTTTWTAAAA-ATTT-TATT--TTT-  617 1611666 AGGGAGGGAGR-GGGGGGGAAAAGA-GAGAA-  618 1611710 GTTTGTTTGGKTTTTTTT-GGGGTG-T-T-GT  619 1611901 TTKTTTTTTTTTTTTTTTTTTT-KTTGTT-TT  620 1611921 CTTTCTTTCCCTTTTTTTTCCCCT-TTCTCCT  621 1612042 A-GGAGG-AGRGGGGGG-GAAAAGARGA--AG  622 1612060 T-GGTGGG-GK-GGGGG-GTTTTGTK-T--TG  623 1612073 A-ATATATAAA-AAAAA--AAAAAAW-A--A-  624 1612200 ATAWATATAAATAAAAAAT-AAAAAWTAT-A-  625 1612354 AAAAAGAAAAAGAAAAAAGAAAAAARGAGAAG  626 1612360 AAAAACAAAAACAAAAAACAAAAAAACAC-AC  627 1612711 TT-WTW-WTTTTTTTTW-TTTTTTTTTT-TTT  628 1612712 TT-YTC-YTTTTTTTTY-TTTTTTTTTT-TTT  629 1612720 GC-SGG-GGCG-GS-SG-GGGGGGGSGG-GG-  630 1612721 CACMCC-CCAC-CM-MC-CCCCCCCMCC-CC-  631 1612760 TAAATA-ATAWAAA-AA-ATTTTATWATATTA  632 1613279 CCCCC--MC-CCCC-CMCMCCCCCCCCCC-C-  633 1613280 AAAAA--WA-AAAA-AWAWAAAAAAAAAA-AT  634 1613290 AAAWAAAAAAATWW-AAAAAAAAAAATA--AA  635 1613292 TTTTTTTTTTT-WT-TTTTTTTTTTT-T--TT  636 1613314 GGGRGGGGGGGGAA-GAAGGGGGGGGGGGGGG  637 1613355 GTGKGGGGGGGGGGGG-GGGGGGGGGGGGGGG  638 1613593 CTCTCC-CC-CCCCCCCCCCCCCCCCCCCCCC  639 1613850 TTTTTATWTTTATTTTTTATTTTTTWTTATTT  640 1614075 CCCMCCCCCCCCAAACAACCCCCACCCCCCCC  641 1614423 TGGGTTGG-GKTGGGGGGTTTTTGTTGTTTTG  642 1614447 GGGGGTGGGGGTGGGGGGTGGGGGGKGG-GGG  643 1614490 AAAAAWAAAAA-AAAA-ATAAA-AAAAA-AAA  644 1614715 T-WTTTT-WTTTTW-TTTTTTTTATTTTTTTT  645 1614758 GCCCGGGCGCSGCCCCCCGGGGGGGGCGGGGC  646 1614819 AGGG-AAGA-RAGGGGG-AAAAAGAAGAAAAG  647 1615080 AGGGAAGAAGRAGGGGGGAAAAAAAAGAAAAG  648 1615669 AAA-A---R-R--RG-A--AAAAAAA-A-A-A  649 1615670 GAG-R---G-R--RA-G--GGGGAGG-G-G-G  650 1615672 AGA-A---A-A--AA-A--AAAAGAA-A-A-A  651 1615675 AGA-A---A-A-GAA-A--AAAAGAA-A-A-A  652 1615684 TGTGTGG-T-T-GTTTT--TTTTGTT-T-T-T  653 1615728 AGAGAGGAAAAGGAAAAAGAAAAGAAAA-AAA  654 1615729 TCTCTCCTTTTCCTTTTTCTTTTCTTTT-TTT  655 1615738 G-GA-AAGGGG-AGGGGGAGGGGAGRGG-GGG  656 1615882 AGARAGG-AGAGGAAAAAGAAAAGA-AAGAAA  657 1615940 C-TTCTT-CTYTTTTTTTTCCCC-CCTCTCCT  658 1615996 T-T-TC--T-T-CTTTTT-TTTT-T-T--TTT  659 1615997 T-T-TA--T-T-ATTTTT-TTTT-T-T--TTT  660 1616062 T-TTTGTTTTTGGTTT-TGTTTTTT-TTGTT-  661 1616174 TCTC-CCCTCTCCTTTTTCTTTTCTYTTCTTT  662 1616203 C-CA-AA-CACAACCCCCACCCCACACCACCC  663 1616335 T-TCT-Y-T-TTTTTTT--TTTT-TTTTCTT-  664 1616336 A-AAA-M-A-AMCAAAA--AAAA-AAAAAAA-  665 1616538 TCTCTCCCTCTCCTTTTTCTTTTCTYTTCTTT  666 1616691 GA-AGAAAGARAAAAAA-AGGGGAGAAGA-GA  667 1617198 AGGGAGGGAGRGGGGGGGGAAA---RGAGAAG  668 1617696 AGRRAG-GAGAGGAAGAAGAAAA-AGGAGAAG  669 1617770 CTTTCTTTCTYTTTTTTTTCCCCTCYTCTCCT  670 1618051 ATTTATTTATWTTTTTTTTAAAATAWTAT-AT  671 1618090 AAAWA-AWAWATWAA-AAAAAAATAA-AAAA-  672 1618231 CGSGCC-CCCCCCCCGCCCCCCCG-CG-CCCG  673 1618254 A-WTAAAAAAAAAAATAAAAAAATAATAAAAT  674 1618273 TCTCTTTTTTTTTTTTTTTTTT-TTTTTTTTT  675 1618347 T-TTTT--T-T-TC-TTC-TTTTT-TTTTTTT  676 1618372 C-TCCC--C-C-TC--CC-CCCC----C-Y-C  677 1618374 T-GTT---T-T-GK--TT-TTTT----T-K-T  678 1618376 A-ARAA--A-AAAA-AA--AAAA----A-A-G  679 1618456 CTT-CTT-CTTTTT-TTTTCCCCTCCTCTCC-  680 1618461 CTC-CCC-CCCCCC-TCCCCCCC-CC-CCCC-  681 1618502 C-YYCC-TCCCCCCCTCYCCCCCTCCTCCCCT  682 1618804 GGGRGGGGG-GGGAAGGAGGGGGGGGGGGGGG  683 1618913 CCMCCCCCCCCCCC-ACCCCCCCACCACCCCA  684 1618914 AAWAAAAAAAAAAA-TAAAAAAATAATAAAAT  685 1619145 AAGAAAGGAGRAAAAGG--AAAAGAAGAAAAG  686 1619316 A--C----A----C-C---AAA-C-A-A-AA-  687 1619317 C--G----C--G-C-G---CCC-GGC-CGCC-  688 1619428 T-WTTTTTTTTTTTTAT--TTTTATTATTTT-  689 1619605 AAWAAAATATWAAAAATAAAAAAAAAAAAAAA  690 1619793 AGGGGGGGAGRGGGGGGGGAAAAGGRGAGAAG  691 1619889 CCCT-C-CC-YC-CCTC-CCCCCCC--CCCCC  692 1619893 TTTA-T-TT-WT-T-AT-TTTTT-T--T-TTA  693 1619897 T-ATTA--T-T-TA-TT--TTTT-A--T-TTT  694 1619898 A-TAAW--A-A-AT-AA--AAAA----A-AAA  695 1619989 GG-GAAGGGGG-AG-GGGAGGGGGAA-GAGG-  696 1619991 AC-CCCCCACA-CC-CCCCAAAACCC-ACAA-  697 1620043 CCYCCCCT---CCC-CTCCCC-CCCC-CCCCC  698 1620056 CCCCTTCCC--TTC-CCCTCCCCCTC--TCCC  699 1620095 TTT-TTA-T---WT---A-TTTT----TWTTT  700 1620101 AAAT-AT-A--AAWT--T-AAAA----AAAAA  701 1620103 ATTA-AA-A--AAWA--A-AAAA-A--AAAA-  702 1620104 TAAT-WT-T--TWWT--T-TTTT-A--TWTT-  703 1620185 CAMAAAACMCCAAAA-CAACCCCAAAACACC-  704 1620249 GCCCCCCCGC--CCCCCCCGGGGCCS-GCGGC  705 1620527 TTTYTTTTTTTTTCCTTCTTTTTTTT-TTT-T  706 1620544 CCMCCCCACCMCCCCCACCCCCCCCC-CCC-C  707 1620585 TCCCCC-CTCYCCCCCCCCTTTTCCYCTCT-C  708 1620728 TTT--T-T--T-TG--KT-TTTTTTT-T-T--  709 1620739 T-T--G-----GGT-TTT-TTTT-GK-T--TT  710 1620813 TGTGTTGTTTTTTTTTTT-TTTTTTTTTTTTT  711 1620946 ATTT-A--A-AAAW---AAAAAAAAA-A-AA-  712 1620954 TAWW-A----TAAW-TATATTTTTAW-T-TTT  713 1620955 TAAA-A----TAAW-AATATTTTTAW-T-TTA  714 1621237 A--TTTT----TT--T----A---TTTA---T  715 1621391 GGGKGGGGGG-GGTTGG-GGGGGGGGGGGGGG  716 1621467 CCACAACCCCCAACCACCA-CCCAAMACACCA  717 1621759 CCCCCCC-C-CCCA-CCACCCCCCCCCCC-CC  718 1621770 AAAAAAAAAAAAAT-AATAAAAAAAAAAAAAA  719 1621799 AAAAAAAAAAAAAT-AATAAAAAAAAAAAAAA  720 1621800 CCCCCCCCCCCCCA-CCA-CCCCCCCCCCCCC  721 1621931 TTTTCCT-T-TCCTTTTTCTTTTTCYTTCTTT  722 1622029 A--A-W-AAAA--AAW-AAAAAA-AA-A-AAA  723 1622034 A-TA-T--ATATTA-T-ATAAAA--ATA-AAT  724 1622108 A-TTTT--A-WTTTTTTTTAAAATTTTATAAT  725 1622131 G-ARAA--G-RAAGG-AGAGGGGAARAGAGGA  726 1622144 C-TYTT--CTYTTCC--CTCCCCTTYTC-CCT  727 1622152 TAAWAA---AWAATTA-TATTTTAAWAT-TTA  728 1622535 CCTYTT-TCCCTTTTTT-TC-CCCTYTCTCCT  729 1622598 T-TWTTTTTTTTTAATTATTTTTTTTTTTTTT  730 1622610 A-GRGGAGAAAGGGGGGGGAAAAAGAGAGAAG  731 1622623 TTTTTTTTTTTTTGGTT-TTTTTTTTTTTTTT  732 1622630 TTAWAATATTTAAAAAA-ATTTTTATATATTA  733 1622659 ATAWAATAATAAAAAAA-AAAAATAAAAAAAA  734 1622728 CCACAACAC-CAAA-AA-ACCCCCAMACACCA  735 1622766 TATATTATTTTTTTTTT-TTTTTTTTTTTTTT  736 1622876 GGRGGGGGGGGGGGGAG-GGGGGGGGAGGGGA  737 1622961 TTCTTTTTTTTTTTTCT-TTTTTTTTCTTTTC  738 1623024 TCCCCCCCTCYCCTTCC-CTTTTCCYCTCTTC  739 1623076 GGGGGGGGGG-GGK-GGTGGGGGGGGGGGGGG  740 1623155 TT-TTTTTTTTTTTTATTTTTTTTTT-TTTTW  741 1623157 TA-ATTATTATTTTT-TTTTTTTATT-TTTTT  742 1623183 CTCTCCTCCTCCCCC-CCCCCCCTCCCCCCCC  743 1623346 AGARAA-AA-AAAAAAAAAAAAARAAAAAAAA  744 1623426 CTCYCCTCCTCCCCCCCCCCCCCTCCCCCCCC  745 1623482 AGARAAGAAGAAAAAAAAAAAAAGAA-AAAAA  746 1623619 GGGG-T-GGGGTTGGGGGTGGGGG-GGGTGGG  747 1623625 AGARGG--AGAGGAAAAAGAAAAG--AAGAAA  748 1623626 CTCTAA--CTCAACCCCCACCCCT-MCC-CCC  749 1623789 TTCTTTTCT-YTTTTCCTTTTTTTTTCTTTTC  750 1623925 TTTYCCTTTTTCCT-TTTCTTTTTCYTTCTTT  751 1624123 GAGAGG--GAGGGGGGGGGGGG-GGGGGGGGG  752 1624435 TCTYTTCT-TTTTTT-TTTTTTTTTTTTTTTT  753 1624569 GTGK-GTTGTGGGGGGGTGGGGGGGGGGGGGG  754 1624739 GGGGGGGG--GGGAAGA-GGGGGGGGG-GGGG  755 1624817 AAAATT-AAAA-TA-AA-T-AAAAT-A-TAAA  756 1625263 TCCC-CCCTCYC-CCCCCCTTTTCCYCTCTT-  757 1625295 A-TA-T-AA-AT---TA--AAAAATA-A-AA-  758 1625296 A-TA-T-AA-AT---TA--AAAAATA-A-AA-  759 1625300 A------AA-W-----T--AAAAT-A-A-AA-  760 1625304 A------AA-W--------AAAAT-A-A-AA-  761 1625331 T----C-TT-T--CC-C--TTT-CCT-T-TT-  762 1625346 CT---T-TC-C--TT-T--CCCCTTC-C-CC-  763 1625392 T-GG-GG-T-T--------TTTT-G-G-G-TG  764 1625409 TCCCCCC-T-T---CC--CTTTT-CTCTCTTC  765 1625424 GAGAGGAAG-G---AG-AGGGGG-GGGGGGGG  766 1625443 CCYCCCCCC-CC-CCTCC-CCCCCCCTCCCCT  767 1625454 GAAAAA-AG-RA-AAAAA-GGGGAARAGAGGA  768 1625472 GGGGAA-GG-GAAG-AGG-GGGGGARAGAGG-  769 1625548 G-------G---AA-----GGGG----G-GG-  770 1625586 GAA----GG-G-GG-----GGGG--G-G-GG-  771 1625587 TGG----TT-T-TT-----TTTT--T-T-TT-  772 1625588 ATT----AA-A-AA-----AAAA--A-A-AA-  773 1625599 CCY---C-C-C-C--TC-CCCCC--C---CC-  774 1625658 C-YT-CT-CTCCCTTCTTCCCCCTCCC-CCCC  775 1625660 C-AA-AA-CACAAAAAAAACCCCAAMA-ACCA  776 1625677 T-CC-CCCTCTCCCCCCC-TTTTCCYC-CTTC  777 1625693 AG-G-GGGAGAGGGGGGG-AAAAGGRG-GAAG  778 1625694 CT-T-CTCCTCCCCCCCC-CCCCCCCC-CCCC  779 1625725 CCAC-ACCCCCAAC-ACC-CCCCC-MACACCA  780 1625788 CT-T--TTCTY-TTT-TT-CCCCT---C-CC-  781 1625833 AA-W-TA-A-A--A-TAW-AAAAA-WTA-AAT  782 1625895 AGAG-AG-AGAAAGGAGG-AAAAG-AA-AAAA  783 1625923 AGAG-AGGAGAAAAAAAAAAAAAAAAAAAAAA  784 1625924 TCTC-TCCTCTTTTTTTTTTTTTTTTTTTTTT  785 1626140 CCACAACACCCAACCACCACCCCCAMACACCA  786 1626248 T----C-CT-TTT--TT--TT-TT-T-TTTT-  787 1626250 C----T-TC-C-C---C--CC-CC-C-CCCC-  788 1626252 A----A-AA-A-T---A--AA-AA-A-ATAA-  789 1626253 T----T-TT-T-A--TT--TT-TT-T-TATT-  790 1626254 G----G-GG-A----GA--GG-GA-G-G-GG-  791 1626263 T----TGTT-TTT--TTG-TT-TTTT-TTTT-  792 1626278 GAG--GAGG-GGGAAGGAGGG-GGGG-GGG-G  793 1626298 CCTCTTCCCCCTTCCTCCTCCCCCTC-CT-CT  794 1626400 ACACAACAA-AAACCAA-AAAAAAAAAAAA-A  795 1626505 CCYCCCCC-CCCCCCTCCCCCCCC-CTCCC-T  796 1626585 AAAAAAATAAA-AAAATAAAAAAAAAAAAAAA  797 1626676 TCYYCCCCTCTCCTTCCTCTTTTTCTCTCTTC  798 1626838 GGGGGG-AGGRGGGGGG-GGGGGGGGGGGGGG  799 1626935 ATTWTTTTATATTAATTATAAAAATWTATAAT  800 1626986 TATWA-ATTAT-ATTTAA-TTTTTATTTATTT  801 1627040 AAAAAWAA-AATWAAAAAWAAAAAWWAAAAAA  802 1627079 GTGTGGT-GTGGGGGGGGGGGGG-GG--GGGG  803 1627098 TTTTTY--TTTTTTTTTTTTTT--TYT-Y-TT  804 1627135 GGGG---GG-GCCGGGGG-GGGGG--G--G-G  805 1627136 TTTT---TT-T-CTTTTT-TTTTT--T--T-T  806 1627245 A----A--A--AAAAT----AAA----AA--T  807 1627248 T----T--T---TTTA----TTT----TT--A  808 1627489 T-TA-TA-TATTTTTTATTTTTTATTTTTTTT  809 1627535 TA-AAA--TAWAA-T--TATTTT-A--TA-T-  810 1627607 ACCMCC-CACCCCAAC-ACAAAACCMCACAA-  811 1627619 TTGTTT-TTTTTTTTG-TTTTTTTTTGTTTT-  812 1627637 AGARAA-AAGAAAA-A-AAAAAAGAAAAAAAA  813 1627649 CCTCCCCCCCCCCCCT-CCCCCCCCCTCCCCT  814 1627669 GGCGGGG-GGGGGGGC---GGGGGG-CGGGGC  815 1627686 AAAAATA-A-AAAAAA--A-AA-ATAAAW--A  816 1627687 TTTTTAT-T-TTTTTT--T-TT-TATTTW--T  817 1627688 AAAAATA-A-AAAAAA--A-AA-ATAAAW--A  818 1627771 TGGKGG-GTGKGGTTGGTGTTTTG-GGTGTTG  819 1627780 CCCCAA-CCCCAACCCC-ACCCCC--CCACCC  820 1627783 GAARAA-AGARAAGGAA--GGGGA--AGAGGA  821 1627802 GAGAGG-GGGGGGGGGG-GGGGGGGGGGGGGG  822 1627934 T-TT---TTTTT-T-TTTCTTTT-TT-TTTT-  823 1628083 AATATT-TAATTTA-T-ATAAAATTWTAT-AT  824 1628315 TT-TTTCT-CTTTTTTCTTTTTTCTTTTTTTT  825 1628562 GGGGCC-GG-GCCGGGGGCGGGGGCSGGCGGG  826 1628644 A--AAACC--MAAAACCA-AAAACAACAAAAC  827 1628651 T--TTTAA--WTTTTAAT-TTTTATTATTTTA  828 1628760 TTCTTT-CT-YTTTT-CTTTTTTCT-CTTTTC  829 1628782 AA-AAW--A-A-WAA-AAAAAAAA-TAAAAA-  830 1628793 TT-TTT--T-T-TTTA-TTTTT---TAT-W--  831 1628955 TTGT-T--T--TTTTGGTTTTT-KTTTT-T--  832 1628956 TTTT-T--T--TTTTTTTTTTT-KTTKT-TT-  833 1628957 GGGG-G--G--TKGGGGGGGGG-GGGKG-GG-  834 1629663 C-TC-C---CCCCCC-C-CCCCT-CC-CCCT-  835 1629665 T-AT-T---TTTTTT-T-TTTT---T-T-TAT  836 1629698 C--C-C--CCCCCCCA-CCCCCAA-CACCC-A  837 1629751 CCTCCC-TCTCCCCCTTCCCCCTTCCTCC-TT  838 1629813 A-GAAAAGAARAAA-GAAAAAAGGAA-AAAGG  839 1629885 TTGTTT--TTTTTTTGTTTTTT--TTGTTT-G  840 1629924 TTWTTT--TTTTTTTTATTTTT-TT-TTTTTT  841 1629966 GGAGGG--GGR-GG-AGG-GGG-AGG-GG-AA  842 1629972 CC-CCC--CCCCCC-TCCCCCC-TCC-CC-TT  843 1629986 GG-GGGA-GGGGGG-AGGGGGG--GG-GGGAA  844 1630016 A-GAAAA-AAAAAAAGAAAAAA--AA-AAA--  845 1630038 A-AAAAGAAGAAAAAAGAAAAA-AAAAAAAAA  846 1630042 T--TTTTCTTYTTTTCTTTTTT-CTTCTTTCC  847 1630059 TT-TTTTCTTYTTTTCTTTTTT-CTTCTTTCC  848 1630073 GG-GGG-AGGAGGGGAGG-GGG-AGGAGGGAA  849 1630087 AAAATT-AA-ATTAAA-A-AAA-ATTAATAAA  850 1630090 GGAGGG-AG-RGGGGA-G-GGG-AGGAGGGAA  851 1630106 TTCTTTT-T-TTTTTCTT-TTT-CTTCTT-CC  852 1630132 TT-TTTT-TTYTTTT-TT-TTT-CTT-TTTC-  853 1630139 GG-GGGG-GGRGGGG-GG-GGG-AGG-GGGA-  854 1630142 CC-CCCC-CCYCCCC-CC-CCC-TCC-CCCT-  855 1630154 GGAGGGG-GGR-GGG-GG-GGG-AGGAGGGA-  856 1630196 A-CAAAA-AAAAAAACAAAAAAC-AA-AAAC-  857 1630198 T-ATTTT-TTTTTTTATTTTTTA-TT-TTTA-  858 1630217 CCTCCCC-CCCCCCCTCCCCCCT-CC-CCCT-  859 1630219 AA-AAAA-AAAAAAAG-AAAAAG-AA-AAAG-  860 1630225 CC-CCCC-CCCCCCCT-CCCCCT-CC-CCCT-  861 1630284 TTATTTTAT-ATTTTATT-TTT-A-TAT-TAA  862 1630303 AAAAAW-AA-A-WAAAAA-AAA-A-AAA-AAA  863 1630311 AA-AAM-AA-A-AAAA-A-AAA-A-MAA-AAA  864 1630325 TT-TTT-ATTTTTTTTTTTTTT-TTTAT-T-T  865 1630357 C-TCCCC-CCYCCCCTCCCCCCTTCCTCCCTT  866 1630376 CCGCCCC-CCSCCCCGCCCCCCGGCCGCCCGG  867 1630447 TT---G--T-TGGT---TGTTT---T-T-T--  868 1630475 CC-CAAAACAM-AAA-AA-CCC---M-CACA-  869 1630513 AA-AAA--AARAAAA-AAAAAA-GAA-AAAG-  870 1630515 CC-CCC--CCYCCCC-CCCCCC-TCC-CCCT-  871 1630608 TT-TTTTATTTTTTTAT-TTTT--TT-TTTA-  872 1630765 TTWTTT--TTATTTTTTTTTTTAWTTATTT--  873 1630766 TTATTT-ATTATTTTAATTTTTAATT-TTT--  874 1630870 CCACCCCAC-ACCC-AACCCCCAACCACCC-A  875 1631079 GGGGGGGGGGGGGGGGG-GGG-AAGGGGGGAG  876 1631156 GGCGGGCCGCSGGGGCCGGGGGCCGGCGGGCC  877 1631191 AAWAAAATA-AAAAA-AAAWAA-TAAWAAA--  878 1631193 CCMCCCCAC-CCCCC-CCCCCC--CCMCCC--  879 1631316 TTATTTTATTW-TTTAATTTTTAA-TATTTAA  880 1631449 CCCCCCTCCTCCCCCCCCCCCCCCCCCCCCCC  881 1631761 GGTGGGTTGTGGGGGTTGGGGGTTGGTGGGTT  882 1631765 AATAAAATAAAAAAATTAAAAATTAATAAATT  883 1632488 TTCTTTTCTTYTTTTCTTTTTTCCTTCTTTCC  884 1633118 TTCTTTTCTTYTTTTCCTTTTTCCTTCTTTCC  885 1633465 AAGAAAAGAARAAAAG-AAAAAGGAARAAAGG  886 1633629 TTTTTTA-TATTTTTTTTTTTTTTTTTTTTTT  887 1633840 GGGGGGAGGAGGGGGGGGGGGGGGGGGGGGGG  888 1633948 GGAGGGAAG-RGGGGAAGGGGGAAGGAGGG-A  889 1633983 TTCTTTCCT-YTTTTCT-TT-TCCTTCTTTCC  890 1634118 GGAGGGAAGARGGGGAAGGGGGAAGGAGG-AA  891 1634206 TT-TTTTGTTTTTTT--TTTTT--TT-TTTGG  892 1634213 GG-GGG--GGGGGGK--GGGGG--GG-GG-TT  893 1634260 CCTCCCTTCTYCCCCTTCCCC-TTCCTCCCTT  894 1634452 TTTTCCCTT-TCCCCTTCCTTTTTCYTTCTTT  895 1634453 GGGGAAAGG-GAAAAGAAAGGGGGARGGAGGG  896 1634537 TTW----AT-W----TA--TTTAA-TTT--AT  897 1634543 TTTY-C-TT-T-CC-TTC-TTTTT-YTT--TT  898 1634611 AAAW-T-AA-A-TT-A-T-AAAAATWAA-AAA  899 1634612 TTTW-A-TT-T-AA-T-A-TTTTTAWTT-TTT  900 1634643 CCCC-TTCC-C----C-T-CCCCC-CCC-CCC  901 1634649 CCCC-TCCC-C-TT-C-TTCCCCC-CCC-CCC  902 1634854 AAARGGAAA-AGGGGAAGGAAAAAGRAAGAAA  903 1634907 AAAATTT-ATATTTT-TT-AAAAATWAATAAA  904 1634974 A-AAGGG-AGAGGGGAGGGAAAAAGRA-GAAA  905 1635001 CCCCCCTCCTCCCCCCTCCCCCCCCCC-CCCC  906 1635093 A-AAAAG-AGAAAAAAAAAAAAAA--AAAAAA  907 1635121 C-CCTT--C-CTTTTCTTTCCC-C-YCCTCCC  908 1635161 T-TTCCT-TTTC-CCTTCCTTTTTCYTTCTTT  909 1635172 T-TTA-T-TTT-TA-TTAATTTTTTTTTATTT  910 1635188 A-AA-TA-AAATTT-AA--AAAAATWAA-AAA  911 1635269 G-GKTTG-GGGTTTTG-TTGG-GGTTGGTGGG  912 1635378 C-CC-T-C--CTTT-C-T-C--CCTTCCTCC-  913 1635479 A-AA---AA-AC---A----A-AA--AA-AA-  914 1635553 CCCSGGGCCGCGGGGCGGGCC-CCGSCCGCCC  915 1635588 A-AAAATA-TAAAAAATAAAA-AAAAAAAAAA  916 1635621 A-AACCMAAAACCCCAACCAAAAACCAAC-AA  917 1635628 T-TTGGTTTTTGGGGTTGGTTTTTGGTTG-TT  918 1635716 AAAA--AAAAAT-T-AA--AAAAA-WAA-AAA  919 1635908 CCCC-C-CC-CYYC-Y-CTCCC-YCCCCCC--  920 1635913 TTTT-W--T-TAWT-T--ATTT-TTTTTWT--  921 1635916 TTTT-W--T-TTWW-T--TTTT-TATTTWT--  922 1635919 AWAA-W----AAAT-A--AAAAAW-ATAAA--  923 1636172 TTCYCCTCTCYCCCCCCCCTTTCCCYCTCTCC  924 1636305 CCCCCCTCCCCCCC-CCCCCCCCCCCCCCCCC  925 1636413 TTTYTTTTTTTTTCCTTCTTTTTTTTTTTTTT  926 1638089 CCCCCTCCCCC-CT-CCCCCCCCC-CCCYCCC  927 1638581 AAAA-AAAA-AAWA--A-WAAAAW-AWAAAA-  928 1638591 CCTC-CY-C-CYCYC-T-CCCCYC-CYC-C-C  929 1638593 TTCT-TY-T-TTTTT---TTTTYT-TYT-T-T  930 1638641 CCYC-TCCCCYTTTTC-TTCC-CCTYCCTCCC  931 1639354 AAAAAATAAAAATAAATAAAAAAAAAAA-AAA  932 1639358 TTWTTTAATTTTTTTATTTTTTAATTAT-TAA  933 1639379 CCTYTTTTCTYTTTTTTTTCCCTT-YTC-CTT  934 1639405 T-ATAA-ATA-AAAAAAA-TTTAA-WAT-T-A  935 1639422 T--T----T-----AW---TTTA--W---T--  936 1639423 T--T----T------T---TTTA--W---T--  937 1639425 T--T-A--T------A-A-TTTT--T---T--  938 1639589 CCGCGGGGCGGGCGGGCGGCCCGG-SGCGCGG  939 1639649 T--T-A--T--AA--AA--TTTA--T-TAT--  940 1639650 T--T-C--T--CT--CT--TTTC--T-TCT--  941 1639656 G--GA-A-G--AG-AAG-AGGG---G-GAGAA  942 1639658 T--TCCC-T--CT-CCT-CTTT---T-TCTCC  943 1639685 AAWAAAATAAAAAAATAAAAAATTAATAAATT  944 1639720 CCCCCCCCCCCCGCCCGCC-CCCCCCCCCCCC  945 1640319 TTTTAATATAWAAA-T--ATTTTTATTTATTT  946 1640338 AAAACCCCACACCC-AC-CAAAAAC-AACAAA  947 1640347 CCCCTTCTCTYTTT-CT-TCCCCCTTCCTCCC  948 1640404 CCCCAACACAMAAAACAAACCCCCAMCCACCC  949 1640483 CCMMAAAACAMAAAACAAACCCCCAMCCACCC  950 1641130 TTTKGG-GTGK-TGGTGGGTTTTTGKTTGTTT  951 1641172 TTTWAA-ATAWTTAATTAATTTTTAWTTATTT  952 1641442 AAAAGGGGA-AAAG-AA-GAAAAAGRAAGAAA  953 1641449 CCCC-CC-C-CCCCACC-CCCCCCMCCCACCC  954 1641451 TTTT-AATT-TTTTTTT-TTTTTTTWTTTTTT  955 1641714 CCYYTTCTCTTCCYTCCTTCCCCCTCCCTCCC  956 1642236 TTTTCCCCTCY-TCCTT-CTTTTTCYTTCTTT  957 1642267 CCYYTTTTCTYCCTTCC-TCCCCCTCCCTCCC  958 1642307 CCYYTTTTCTYCCTTCC-TCCCCCTYCCTCCC  959 1642711 TTTTGKTKTGK-TGGTTGGTTT-TGKTTGTTT  960 1643324 CCYCTTT-CTTCCTTCC-TCCCCCTYCCTCCC  961 1643682 TYTTTTTTYTTTTYTTYTTTTTTT-TTTTTTT  962 1643963 GGGRAAGGGGGGGAAGGAAGGGGGAAGGAGGG  963 1644011 CCTYTTTTCTTTTTTTTTTCCCTTTYTCTCTT  964 1644076 GGGSCCCCGCSGGCCGGCCGGGGGCSGGC-GG  965 1644328 TTTYCCTCTCYTTC-TTCC-TT-TCYTTCTTT  966 1644511 TTTT--T-T-T-CT-TCT-TTTTT-TTTTT-T  967 1644513 GGGG-TG-G-KGGT-GGT-GGGGG-GGGTG-G  968 1644525 TTTTTTCTT-TTTT-TTTTTTTTT-T-TTTTT  969 1644537 AAWAAAATA-WAAAAAAAAAAAAAAAAAAAAA  970 1644750 GGGGGGGGGGGTTGGTTGGGGGGGGGTGGGGT  971 1645218 AATAAATAA-ATTAATT-AAAAATAA-AAAAT  972 1645228 GGGKTTGGG-GGGTTGG-TGGG-GTTGGTGGG  973 1645334 CYCCTTCCCCCCCTTCCT-CCCCCTYC-TCCC  974 1645582 GGCGGGCGGGGCCGGCC-GGGGGGGGCGGSCC  975 1645815 AAGAAAAAA--GGAAGGAAAAAAAAAGAAAAG  976 1646008 AACMCCCCM-ACCCC-CC-AAAAACMCACAAC  977 1646226 AAAAAA-MA-AAA--CA--AAAAA-AAAA-AA  978 1646229 CCCCCC-CC-CYY--CC-CCCCCC-C-CC-C-  979 1646564 TTTYTTTTTTTTTC-TTCTTTTTTTTTTTTTT  980 1646600 AAWW-ATAA-ATATAW-ATAAAAAAATAAAA-  981 1646602 GGRR-GAGG-GAG-GR-GAGGGGGGGAGGGG-  982 1646890 AAWAAAATAAWAAAAAA-AAAAAAAAAAAAA-  983 1647863 GGGSGSGGG-GGGS-GSGGGGGGSG-SGSGGC  984 1648561 AAMACCAAAAACCCCCC-CAAAACCCCACAAC  985 1649069 GGGGGGTGGGGKGGGGGGG-GGGT-GGGG-GG  986 1649189 AATWTTTTATWTTT-TTTTAAAATTATATAAT  987 1649194 TTAWAAAATAWAAA-AAAAWTTTAATATATTA  988 1649212 AAAAAACAAAAAAA-AAAAAAAACAAAAAAAA  989 1649292 A-GAAAAAAAAGGA--G-AAAAAAAAGAAAAG  990 1649335 T--TTWT-TTTWTT--TTTTTT-A-T--TT-T  991 1649343 A-AAAA-AAAAWW--AWAAAAAAAAAA-AA-A  992 1649385 AAA-AAGA-AAAAA-AAAAAAAAGAAAAAAAA  993 1649892 TTATTTATTTWAATTAATTTTTTTTTATTTTA  994 1649975 AAGAAAAAAARGGAAGG-AAAAARAAGAAAAG  995 1650007 AAGAAAAAAAAG-AAGGAAAAAAAAAGAAAAG  996 1650035 TTCTTTTTTTYCCTTCCTTTTTTTTTCTTTT-  997 1650053 TTYTTTTTTTTCCTT-CTTTTTTTTTCTTTT-  998 1650057 AARAAAAAAARGGAAGGAAAAAAAAAGAAAA-  999 1650062 AARAAAAAAARGGAAGGAAAAAAAAAGAAAA- 1000 1650140 GGTGGGTGGGKTTGGTTGGGGGGGGGTGGG-T 1001 1650162 T-TTTTTTTTTTTTTTTTTTTTWTTTTTTTAT 1002 1650201 G-GGGGAGGGGGGG-GG-GGGGGGGGGGGGGG 1003 1650312 GGARAAAGGGRAAAAAAAAG-GGGAR-GAGGA 1004 1650501 TTTTTTATTTATTTTTTTTTTTTTTTTTTTTT 1005 1650892 TTTT-AT-T-T-T--T--ATTTTTT--TWT-- 1006 1651006 TTYTTTTTTC--TTTC-T-TTTTT--CTTT-C 1007 1651019 TTWTA-T-TAT--AAATA-TTTTT--AT-T-A 1008 1651075 AAAAGGA-AAAAAGGAAGGAAAAAGRAAGAAA 1009 1651085 CCCCCCC-CCCAACCCACCCCCCCCCCCCCCC 1010 1651266 TTTTTTTCTTTTTTTTT-TTTT-YTTTTTTCT 1011 1651404 AAWAAAATATWAAA-TAAAAAA-A-ATAAATT 1012 1651496 AAAA-AAAAAAGGAAAGAAA-AAAA-AAAAAA 1013 1651574 A--AAAAAAAAWTAA-TAAAAA-AAA-AAAA- 1014 1651575 T--TTTTTTATW-TT-TTTTTT-TTT-TWTT- 1015 1651579 AA-AAAA-AAARGAAAAAAAAA-AAA-AAA-A 1016 1651787 A-AAAAA-AA-GGAAA-A-AAAAAAAAAAAAA 1017 1651950 A-AMCCM-AAM-ACCAA-CAAACACCAACACA 1018 1651977 AAAWTTT-AAW-TTTATT-AAATATWAATA-A 1019 1651991 AAWWTTT-ATT-ATTTAT-AAA-ATWTATA-T 1020 1651993 TTTTTTT-TTT-ATTTAT-TTT-TTTTTTT-T 1021 1651996 TTTWAA--TTW-TAATTA-TTT-TAWTTAT-T 1022 1652011 CCCMAAA-CCCAAAACAAACCCACAMCCACAC 1023 1652098 AAAAAAAAAAACCAAACAAAAAAAAAAAAAAA 1024 1652139 AACAAAAAACAAAAACAAAAAAAAAACAAAAC 1025 1652235 GGCGCCCCGCGCCCCCC-CGGGCGCSCGCGCC 1026 1652253 AAAACCCCAAACCCCAC-CAAACACMAACA-A 1027 1652352 TTTTTTTTTTTTTTTCTTTTTTTTTTCTTTTC 1028 1652357 CCCYTTCCCCCTTTTCTCTCCCCCTYCCTCCC 1029 1652401 TTTTTYCCTTYTTTTTTCTTTTCTTTTTTTCT 1030 1652453 AAGRGGGGAGRGGGGGG--AAAGAGRGAGAGG 1031 1652715 GCGGG-GGSG--SG-GG-CG-SGGG---SGG- 1032 1652723 TTT-T-CCT-TTTT-TT-TTTTC-TT--T-C- 1033 1652747 GGGGGGGAGGGGGG-G--GGGGAGGGGGG-AG 1034 1653494 AAAAAAAAAAAGGAAAGAAAAAAA-AAAAAAA 1035 1653520 CCMCCCCCCCACCCCACACCCCCC-CACCCCA 1036 1653597 GGGGGGGAGGGGGGGGGGGGGGAG--GGGGAG 1037 1653769 CCCCCCCCCCSCCCCCCGCCCCCCCCCCCCCC 1038 1653795 AAAAAAAGAAAAAAAAAAAAAAGAAAAAAAGA 1039 1653979 GGGGGGGTGGGTTGGG--GGGGTGGGGGGGTG 1040 1654008 AAAAAAAAAAATTAAA--AAAA-AAAAAAAAA 1041 1654084 TTTTTTTTTTTGGTTTGTTTTTTTTTTTTTTT 1042 1654113 CCCCCCCCCCC-GCCCGCCCCCCCCCCCCCCC 1043 1654119 GGAGAAAAGAR-AAAAAAA-GGAGARAGAGAA 1044 1654174 GGGGGGGTGGGGGGGGG-GGGG-GGGGGGGTG 1045 1654178 TTTTTTTCTTTTTTTTT-TTTT-TTTTTTTCT 1046 1654234 AAAAAAAAAAAGAAAAR-AAAAAAAA-AAA-G 1047 1654235 AAAAAACAAAA-AAAAM-AAAA-AAA-AAA-- 1048 1654287 AAAAAAT-AAA--AA-T-AAAA-AAA-AAA-T 1049 1654362 TTTTTTTCTTTTTTTTTT-TTTCT-TTTTTCT 1050 1654368 GGAGGGGAGGGAAGGAAGGGGGAG-GAGGGAA 1051 1654467 TTKTTTTTTTTGGTTGG-TTTTTT-TGTTTTG 1052 1654484 AAGAAAGGAAAGGAAGGAAAAAGA-AGAAAGG 1053 1654519 TTATTTTTTTTAATTAATTTTTTTTTATTTTA 1054 1654529 CC-C-CCCCCC--CC--CCCCCCCCCGCCCCG 1055 1654530 TT-T-TTATTT--TT--TTTTTATTTTTTTAT 1056 1654634 GGGGGGGGGG-AAGGGAGGGGGGGGGGGGGGG 1057 1654649 GGGGGGGGGGGAAGGGAGGGGGGGGGGGGGGG 1058 1654795 TTTTTTAATTAAAT-TAATTTTATTTTTTTAT 1059 1654849 CCYCCCTTCCYTTCCTTTCCCCTCCCTCCCTT 1060 1654906 CCCCCCAACCMCCC-CCACCCCMCCCCCCCAC 1061 1655007 CCCCCC-ACCCCCC-CC-CCCCACCCCCCCAC 1062 1655042 GGGGGGGGGGGCCG-GCGGGGGGGGGGGGGGG 1063 1655195 CCCCCCTTCCYCCCCCC-CCCCCCCCCCCCC- 1064 1655349 TTTTTT--TTT-ATT-T-TTTT-TTTATTTTT 1065 1655353 GG-GGGAAGGR-AGG-AAGGGG-GGGAGGGG- 1066 1655408 GGGGGGAAGGGGGGGGGGGGGGGGGGGGGGG- 1067 1655881 TTGTTTTTT-TTTTTGTT-TTTTTTTGTTTTG 1068 1656044 GGGGGGAGGGGAAGGGAGGGGGGGGGGGGGGG 1069 1656188 AARAAAG-AAAAAA-AA--RAAAAAARAAAAA 1070 1656189 GGSGGGC-GGGGGG-GG--GGGGGGGSGGGGG 1071 1656263 GGG-GGCCGGGGGGGGGGGGGGGGGGGGGGGG 1072 1656348 TTATAAAATAAAAA-AAAATTTATATATATAA 1073 1656394 CCYCCC-TCCCTTCCTTTCCCCCCCCTCCCCT 1074 1656622 TTYTTTTTTTTTTTTCTTTTTTTTTTCTTTTC 1075 1656645 TTTTTTTTTTTCCTTTCTTTTTTTTTTTTTT- 1076 1656898 C-CCCCTTCCCCCCC-C-CCCCCCCCC-CCCC 1077 1656979 GGGGGGAGGGGGGGGGGGGGGGGGGGGGGGGG 1078 1657025 AAWAAATT-AWTWAATT-AAAAAAAATAAAA- 1079 1657162 CCCCCCTTCCCCCCCCCCCCCC-CCCCCCCCC 1080 1657319 GGGGGGGGGGGCCG-G---GGGGGGGGGGSG- 1081 1657593 AAAAAAATAAATAAAA-AAAAAAAAA-AAAAA 1082 1657661 AAAAAAATAAATTAAAT-AAAAAAAAAAAAAA 1083 1657803 TTTTTT--TTAAAT-TA-TTTTTTTT-TTTTT 1084 1657814 AAAATT--AAT-TT-A--TAAAAAWWAATAAA 1085 1657857 TTTTTTTC-TTCCTTT--TTTTTTTTTTTTTT 1086 1657867 AAAAAAAGAAAGGAAA-AAAAAAAAAAAAAAA 1087 1658176 CCCCCCCCCC-T-CCCTTCCCCCCCCCCCCC- 1088 1658177 GGGGGGGGGG-TKGGGTTGGGGGGGGGGGGG- 1089 1658205 CCCCCCC-CCCAAC-CACCCC-CCCCCCCCC- 1090 1658219 CCCCCCC-CCTTTCCCTT-CCCCCCCCCCCC- 1091 1658281 TTTT-TTTT-YCCTTTCCT-TTTTTTTT-TTT 1092 1658580 AAAAAAAA-AAAAAATTAAAAAAAA--AAAAT 1093 1658589 AAAAAAACAACAAAAC-CAAAAAAA--AAAA- 1094 1658590 CCCCAACCCCCCCCCC-CAACCCCC---ACC- 1095 1658617 AAAA-AGAAAAGGAAAGAAAAAAAA-AAAAAA 1096 1658632 CCCCCCC-CCCTTCCCTCCCCCCCC-CCCCCC 1097 1658707 GGAGGGA-GGG-AGGAA-GGG-GGGGAGGGG- 1098 1658756 GG-GGGGGGGGTTGGGT-GGGGGGGGGGGGG- 1099 1659101 AAAAAAAAAAAGGAAAGAAAAAAAAAAAAAAA 1100 1659190 AAAAAAAAAAAGGAAAGAAAAAAAAAAAAAAA 1101 1659382 TTTTTWTTTTT-TT--TT-TTTTT-W-TTTT- 1102 1659384 AWWAAAATAAW-AA---T-AAAAA-ATAAAA- 1103 1660414 CCMCCCCACCMAACCAAACCCCCCCCACCCCA 1104 1660604 AAWA---AA-A-AT-WT--AAAAA--AA-AA- 1105 1660606 TTTT---TT-T-AA-TA--TTTTT--TT-TT- 1106 1660618 GGGGT--GG-GTG--G---GGGGG-GGG-GGG 1107 1660621 AAAAG--AA-AGAG-A-A-AAAAA-AAA-AAA 1108 1660668 AAAAGGAAA-AAAG-AAAGAAAAA-RAAGAAA 1109 1660682 TTATAAATT-TTAA-ATAATT-TT-WATATTA 1110 1660704 TT-TTTTTTTTTCTTCT-TTTTTT-TC-TTT- 1111 1660795 CCCCCCWCC-CCCY-CC--CCCCCTC-C--CC 1112 1660796 TTTTTTTTT-TTTY-TT--TTTTTCT-T--TT 1113 1660935 T-YTTTTTTTTT--TCTC-TTTTTTTCTT-TC 1114 1660958 CCMCCCCCCCCCAC-AC--CCCCCCCACCCCA 1115 1660963 TTCTTTTTTTTTCT-CT--TTTTTTTCTTTTC 1116 1661003 CC-CCCC-CCCTTCCTT-CCCCCCCCTCCCC- 1117 1661011 TT-TTTT-TTTTCTTCT-TTTTTTTTCT-TT- 1118 1661015 GG-RAAG-GAGGGAAGG-AGGGGGAGG--GG- 1119 1661025 CC-SGGCCCGCC-GGCC-GCCCCCGCC--CC- 1120 1661066 A-AA-AAAAAAAAAWAAA-AAAAA-AAA-AAA 1121 1661084 T-CT-TTTT-TCC-TCCC-TTTTTTTCT-TTC 1122 1661088 A-GA-GGAA-AGG-GGGG-AAA-AGAGA--AG 1123 1661104 T-CT-CCTT-TCC-CCCC-TTT-TCTCT--TC 1124 1661128 A-GA-GG-A-AGGGGG-G-AAAAAGAGA-AAG 1125 1661129 C-CC-TC-C-C-CTTC-C-CCCCCTCCC-CCC 1126 1661131 G--G-GG-G-GG-GGC-C-GGGGGGGCG-GGC 1127 1661150 C--C-CC-C-CCT----T-CCCCCCC-C-CCT 1128 1661160 T--T---TT-TCC----C-TTTTT-TCT-TTC 1129 1661214 T-GTTTTTTTKGGTT-GG-TTTTT-TGTTTTG 1130 1661239 TTCYCCCTTCC-CCCC-CCTTTTT-TCTCTTC 1131 1661259 CCSSGGGCCGCCCGGCCCGCCCCCGCCCGCCC 1132 1661275 CCYCCC-CCCYCTCCTCTCCCCCCCCTCCCCT 1133 1661328 T-CYCCCTTC-CCCCCCCC-TTTTCTCTCTTC 1134 1661356 A-RAGGGAAGAAAGGAAAG-AAAAG-A-GAAA 1135 1661456 TT---AA-TAT---A----TTT-T-T-TATT- 1136 1661461 AA---AA--AA---AG-G-AAAAA-AGAA--- 1137 1661539 T--TTTTTT-CTCTT-TCTTT-TTTTCTTTTC 1138 1661560 T-ATTTT-T--T-TTAT--TT-TTTT-TTTT- 1139 1661768 CCYCCCCCCCYCTCCTCTCCCCCCCCTCCC-T 1140 1661783 GGRGGGGGGGRAAGGAA-GGGGGGGGAGGGGA 1141 1661971 AAWAAAA-AAWATAATAT-AAAAAAATAAA-T 1142 1662071 CCMCCC--CCCCACCAC-C-CCCCCC--CCC- 1143 1662122 TT-T-T-TTTTTT---T--TTTTT-TATATTT 1144 1662124 AATATT-AATAAA---A--AAAAA-WAAAAAA 1145 1662258 CCYCCCCCCCYCTCCTCTCCCCCCCCTCCCCT 1146 1662283 CCACCCCCCCMCACCACACCCCCCCCACCCCA 1147 1662373 CCCCCCCGCCCGCCCCG-CCCCCCCCCCCCCC 1148 1662399 TT-TTTGKTTTGKTTT--TTTTTTTTKTTTTT 1149 1662401 TT-TTTTTTTKTTTTGT-TTTTTTTTKTTTTT 1150 1662555 GGGGAAGGGAGGGAAGGGAGGGGGAGGGA-GG 1151 1662568 AARAAAAAAA-AGAAGAGAAAAAAAAGAAAAG 1152 1662576 AACAAAAAAA-ACAACACAAAAAAAAC-AAAC 1153 1662653 GGKGGG-GGGKGTGGTG--GGGGGGGTGGGGT 1154 1662656 CCSCCC-CCCSCGCCGC--CCCCCCCGCCCCG 1155 1662666 GG-GGG-GGGSGCGGCG--GGGGGGGCGGGGC 1156 1662672 TT-KGG-TTGTTTGGTT--TTTTTGKTTGTTT 1157 1662692 CC-CCC-CC-YCTCCTCT-CCCCCCCTCCCCT 1158 1662694 TTCTGG-TT-YCCGGCCC-TTTTTGTCTGTTC 1159 1662706 CCCCTT-CC--TCTT-TC-CCCCCTYCC-CCC 1160 1662725 GGCG-G-GG---CGG-GC-GGG-G-G-G-GGC 1161 1662758 GG-G---GG-RGA----A-GGGGG-G-G-GG- 1162 1662766 TTCTCC-CT-YCCC---CCTTTTTCY-T-TT- 1163 1662804 CCTCTT-CC-C--T--C--CCCCCTY-CTCC- 1164 1662841 CC-CTT-CC----T----TCCCCCTY-CTCC- 1165 1662854 TT-T-GTT--T--G----GTTTTTTT-TKTT- 1166 1662858 GG-R--GGG-G--------GGGGGAR-GAGG- 1167 1662879 GG-R-AGAG-GG-A--G--GGGGGAR-GAGG- 1168 1662882 GG-G-GGGG-GT-G--T--GGGKGGG-GGGG- 1169 1662894 AAGR-GGAA-AG-G--G-GAAAAAGG-AGAA- 1170 1662916 GGKGGGGGGGKGTGGTG-GGGGGGGGTGGGGT 1171 1662935 TTCTCCCCTCYCCCCCCCCTTTTTCYCTCTTC 1172 1662980 GGAGGGGGGGR-AGGA-RGGGGGGGGRGGGGA 1173 1662997 CCCCCMCCCCCCCCCC-MCCCCCCCCMCCCCC 1174 1663007 TT-TTTCTTTTCTTTT-TTTTTTTTTTTTTTT 1175 1663014 GG-GGRTGGGGGGGGG-RGGGGGGGGRGGGGG 1176 1663032 GG-GGRAGGGGGAGG--AGGGGGGGGAGGGG- 1177 1663033 CC-CCYCCCCCCCCC--TCCCCCCCCTCCCC- 1178 1663051 GGARAAAGGAG--A-A-AAGGGGGAR-GAGG- 1179 1663064 AAGAAAGAAAAGGA-G-GAAAAAAAR-AAAA- 1180 1663110 GGGGGGGAGGG--GG--AGGGGGGGG-GGGG- 1181 1663143 AAGAGGGGAGA-GGG-G-GAAAAAGR-AGAAG 1182 1663181 CCTYTTTCCTCATTTTA-TCCCCCTYTCTCCT 1183 1663182 GGGGGGGGGGGAGGGGA-GGGGGGGGGGGGGG 1184 1663188 AAGAAAAAAAAAGAAGA-AAAAAAAAGAAAAG 1185 1663219 CC-CCCCCC-CA-C--A--CCCCCCC-C-CC- 1186 1663226 CC-CTTTCC-CT-T-----CCCCCTC-C-CC- 1187 1663237 CC-CC--CC-CA-MC-A-CCCCCCCC-CCCC- 1188 1663250 GGAGGGAGG-GA-GG-A-GGGGGGGGAGGGG- 1189 1663251 CCTCCCCCC-CC-CC-C-CCCCCCCCTCCCC- 1190 1663288 CCSCCCCCCCCCGCCGCGCCCCCCCCGCCCCG 1191 1663298 AAGRGGGAAG-GGG-GGGGAAAAAGAGAGAAG 1192 1663445 GGGGGG-GGGGAGGGGAGGGGGGGGGGGGGGG 1193 1663463 CCCCCC-CCCCTCCCCTCCCCCCCCCCCCCCC 1194 1663479 AAGRGG-AA-AGGGGGGGGAAAAAGR-AGAAG 1195 1663486 GGARAA-GG-GG-A-GG--GGGGGAR-GAGGG 1196 1663501 CC-Y---CC-CC-T--C--CCCCCTY-C-CC- 1197 1663505 CC-CA--CC-CM-C--C--CCCCCCC-C-CC- 1198 1663520 CCCCC-CCCCCT-CC-TC-CC-CC-CCCCCCC 1199 1663534 GGRRAAGGGAGGGAA-GG-GG-GG-GGGAGGR 1200 1663535 TTTTTTCTTTTCTTT-CT-TT-TT-TTTTTTT 1201 1663553 TTYTTT-TTTTCCT---C-TT-TTTT-T-TTC 1202 1663562 CCMCCC-CCCCCAC-A--CCCCCCCCACCCC- 1203 1663564 AARAAA-AAAAGGA-G--AAAAAAAAGAAAA- 1204 1663568 AARRGG-AAGAGAG-A-AGAAAAAGRAAGAA- 1205 1663573 AAARGG-AAAAAAG-A-AGAAAAAGRAAGAA- 1206 1663620 TTCT-C-TT-TC-----C-TTTTT-TCT-TT- 1207 1663628 TT-T-G-TT-TT---GGTGTTTTTGTTT-TT- 1208 1663698 CCYCCCC-CCYCTCCTCTCCCCCCCCTCCCCT 1209 1663719 CCYCCCC--CYCTCCTCTCCCCCCCCTCCCCT 1210 1663724 GGRGAAG--AGGGAA-GGAGGGGGA-GGAGGG 1211 1663731 CCACAA-CCAMAAAA-AAACCCCCAC-CACCA 1212 1663747 TTKTTT-TTTKT-TT-TG-TTTTTTTG-TTTG 1213 1663777 GGRGGGGGGGRA-GGAAA-GGGGGGGAG-GGA 1214 1663798 GG-G--GGG-KG---TG-GGGGGG-GTG-G-T 1215 1663907 CCGSGG-CC-CGGGG-G--CCCCCGSGCGCC- 1216 1663935 AA-AGA-A--AAAGAAAA-AAAAA-A-A-AAA 1217 1663957 AA-ATT-AATAAA-TAAATAAAA-TW-A--AA 1218 1664033 AAGAAAA-AARGGAAGGGAAAAAA-AGAA-AG 1219 1664115 GGARAAAGGARAAAAAA-AGGGGGARAGAGGA 1220 1664472 AAAA-T-AA-A-ATTA-AWAAA-A-AAAAAAA 1221 1664473 TTTT-A-WT-W-TA-T-TTTTT-T-TTTTTTT 1222 1664494 AAW----AA-A-A--TTAAAAAAA--AAAAA- 1223 1664496 TTW---TTT-T-T--AATTTTTTT-TTTTTT- 1224 1664567 AATATTTTATTATTTTATTAAAAATWTATAAT 1225 1665072 AAAAAAAAA-ACAAA-CAAAAAAA-AAAAAAA 1226 1665111 GGGGGGGGGGGTGGG-TGG-GGGGGGGGGGGG 1227 1665167 CCCCCCC-C-CACCCCACCCCCCCCCCCCCC- 1228 1665182 T-AWAAAATAWAAAAAAAATTT-TAWATATT- 1229 1665185 AAAAAAAAAAATAAAATAWAAA-AAAAAAAA- 1230 1665206 AATATTATATWTTTTTTTTAAAAATATATAA- 1231 1665230 AAAAAAAAAAAGAAAAGAAAAAAAAAAAAAAA 1232 1665914 TTTTTT-TTTTATTTTATTTTTTTTTTTTTTT 1233 1665941 AAWAAA-AAAWATAATATAAAAAAAATAAAAT 1234 1666046 TTATTT-TTTT-TTTWT-TTTTTTTT-TTTTA 1235 1666527 TTTTTTATTTTATTTTATTTTTTTTTTTTTTT 1236 1666561 TTCTTTCTTTYCCTTCCCTTTTTTTTCTTTTC 1237 1666912 AAAAAAAAAAAGAA-AG-AAAAAAAAAAAAAA 1238 1667354 TTTT-TCTTTTCTTTTC-TTTTTTTTTT--TT 1239 1667743 TTTTTTATTTTAWTT-A-TTTTTTTTTTTTT- 1240 1667747 GGSGGGGGGGGGSGG-G-GGGGGGGGCGGGGG 1241 1667810 CCCCCCCCCCCTCCCCTCCCCCCCCCCCCCC- 1242 1668348 AAAWTTATAAAAATTAA-TAAAAATWAATAAA 1243 1668441 AAGAAAGAAARGGAAGGGAAAA-AAAGAAAAG 1244 1668887 GGRGGGAGG-RAAGGAA-GGGGGGGGAGGGGA 1245 1668893 CCYCCCCCC-YCTCCTC-CCCCCCCCTCCCCT 1246 1668996 TTGTTTTTTTKTTTTGT-TTTTTTTTGTT-TG 1247 1669034 AA-A---AA-W-WA-A--AAAAAAAAWAAAA- 1248 1669145 AAAATTA-A-AAATTAA-TAAAAATAAA--AA 1249 1669151 TTATTTA-T-TTATTAT-TTTTTTT-AT--TA 1250 1669190 GGRGGGAGGGRAAGGAAAGGGGGGGGAGGGGA 1251 1669348 CCTYTT-TCTYTTTTTTTTCCCCCTYTCTCCT 1252 1669541 TTYTTT-TTT-TCTTCTC-TTTTTTTCTTTTC 1253 1669557 GGGGGG-GGG-GAGG-G-GGGGGG-GAGGGGA 1254 1669564 AAAAAA-AAA-A-AATA-AAAAAA-ATAAAAT 1255 1669584 CCMCCC-CCCCCACCACACCCCCCACACCCCA 1256 1670005 GGARAA-AGAGAAAAAAAAGGGGGAAAGAGGA 1257 1670010 TTTTTT-TTTTCTTTTCTTTTTTTTTTTTTTT 1258 1670034 TTCTTT-TTTTTCTTCTCTTTTTTTTCTTTTC 1259 1670046 TTGTTTGTTTTTGTT-T-TTTTTTTT-TTTTG 1260 1670786 TTCTCC--TC-TCCCCTCCTTTTTCC-TCTTC 1261 1671225 TTCYCCTCTCCTTCCCTTCTTTTTCYCTC-TC 1262 1671483 AAGAGGAGAGRAAGGGAAGAAAAAGRGAGGAG 1263 1671607 TT-T--TAT-T-TA--TT-TTTTT-T-T-TT- 1264 1671644 GGGG--GRG-G-GG-GRGGGGGGGRG-GR-G- 1265 1671646 TTKT--TKT-T-TT-TKTTTTTTTKTGTK-TK 1266 1671655 TTTT-T-TTTT-T-TCT-CTTTTTTTTTTTTT 1267 1672660 CCCCCCCCCCCCACCCCACCCCCCCCCCCCCC 1268 1673096 TTGKGGGGTGKGGGGGGGGTTTTTGKGTGGTG 1269 1673273 AAWWTT-TWTAATTTAAT-AAAAATWAATAAA 1270 1673437 AAAAAAAAAAAAGAAAAGAAAAAAAAAAAAAA 1271 1673454 CCCYTTCTCCCCTTTCCTTCCCCCTYCCTCCC 1272 1673470 AA-AGGAGAAAAAGGAAAGAAAAAGR-AGA-- 1273 1673499 CCCYTTCTCCCCTTTCCTTCCCCCTY-CTCCC 1274 1673512 GGRGGGGGGAGGGGGGG--GGGGGRGGGGGGG 1275 1673576 GGAGGGGGGGAGGGGAG-GGGGGGGGAGGGGA 1276 1674853 CCCCCC-CCCSCCCCCC-CCCCCCCCCCCGCC 1277 1674957 TTTTTTATT-TTTTTTTTTTTTTTTTTTTATT 1278 1674960 CCCCCCTCC-CCCCCCYCCCCCCCCCCCCTCC 1279 1674972 CCCYTTTTC-CCTTTCCTTCCCCCTYCCTTCC 1280 1675064 GG-RAA-AGGGGAAAGGAAGGGGGARGGAAGG 1281 1675070 GG-RAA-AGGGGAAAGGAAGGGGGARGGAAGG 1282 1675285 CCCCCC-CCCCTCCCCTCCCCCCCCCCCCCCC 1283 1675368 AA-A-T--AAAT---A----AAAAAAAA--AA 1284 1675396 AAAA-G-GAAA-G--A----AAAAAAAA--AA 1285 1675397 GGGG-A-AGGG-A--G----GGGGGGGGAAGG 1286 1675405 TTTT-T-TTTTTTTTTT---TTTTTTTTTTTT 1287 1675552 AAGAAAAAAGAAAAAGAAA-AAAAA-GAAAAG 1288 1675709 GGAGGGGGGAGGGGGAGGGGGGGGGGAGGGGA 1289 1675849 AAAMCCACAAAACCCAA-CAAAAACM-ACAAA 1290 1675982 CCTCCCCCCTCCCCCCCCCCCCCCCCCCCCCC 1291 1676018 AACMCCCCACM-CCCA-CCAAAAACMAACCAA 1292 1676087 AAAAAAMAA-A-AAA-AAAAAAAAAACAACAA 1293 1676145 AATTTTTTA-WTTT--TTT-AA-T-A-ATTAT 1294 1676226 AATA-TTAA-A-ATTT--AAAAAA---ATTA- 1295 1676227 TTGT-GGTT-T-TGGG--TTTTTT---TGGT- 1296 1676291 CCTCCCC-C-CCCCCTCCCCCCCC-CTCCCC- 1297 1676587 TTGT-TG-TGKGGK-GG--TTTTT-KGTK-TG 1298 1676704 GGAGA---G-GA-------GGGGG---G--G- 1299 1676705 AACAC---A-AC-------AAAAA---A--A- 1300 1676732 TT-TAAA-T--AA------TTTTT---TA-T- 1301 1676809 T--T-TA----T-T-TAT-TTTTTTTWT--T- 1302 1676925 TTAWAA--TA--AAA--AATTTT--AATAAT- 1303 1676981 TTCTC----C--CC-----T-------T--T- 1304 1677110 C--Y----C---T--T---CC---------CT 1305 1677142 G--K----G-G--T-T---GG--G------GT 1306 1677210 A-GAGG--A-G-G----G--AAA--A---GA- 1307 1677244 TTCYCCCCT-Y-C-CCCC-TTTT--TCTCCT- 1308 1677273 TTGKGGGGTGKGGGGGGGGTTTTTGKGTGGT- 1309 1552671 AGAG-AGGAA-GGG-AG-AGAAGA--AAAAGA    8 (SNAP Stop)

127 SNPs (FP88-S127, light-grey), 21 insertions (FP88-121, light-grey), and 27 deletions (FP88-D27, light-grey), possessed by Forrest, Peking, and PI88788, but not by Essex, confer resistance of Rhg1 to SCN (see e.g., FIG. 4). 6 SNPs (C163208A, C163225G, G164965C, G164968T, G164968C and C164974A) and 4 insertions (A164972AGGT and A164972AGGC) locate within the exons of SNAP, and cause amino acid (AA) changes to the predicted SNAP protein (see e.g., TABLE 7).

TABLE 7 AA (nucleotide) Total AA changes in AA (nucleotide) Gene SNPs/Insertions changes Forrest/Peking changes in PI88788 Glyma18g02353 1 1 1 — Glyma18g02420 1 1 1 — Glyma18g02450 3 3 3 — Glyma18g02520 1 1 — 1 Glyma18g02590 10 9 3 + 2* 4 + 2* (SNAP) D208E (C163225G) Q203K (C163208A) D286Y (G164968T) E285Q (G164965C) D287E* and D286H (G164968C) −288V (A164972AGGT) D287E* and −288A L289I* (C164974A) (A164972AGGC) L289I* (C164974A) Glyma18g02650 1 1 1 — Glyma18g02660 1 1 1 — Glyma18g02681 1 1 1 — Glyma18g02690 3 3 3 — Glyma18g02700 1 1 1 — Glyma18g02720 2 2 — 2 Glyma18g02741 1 1 — 1 Where * indicates that the amino acid changes take place in both Forrest/Peking and PI88788.

Example 7 Tilling Screening and Phenotyping

The following example describes additional evidence for the identification of a missense Forrest SNAP Type III mutation of A111D in SEQ ID NO: 7.

Forrest SNAP Type III missense mutant A111D was screened by TILLING from the newly developed, chemically mutagenized SCN-resistant soybean Forrest population. SNAP in the A111D mutant was sequenced to characterize the identified allele and its subsequent amino acid changes within the predicted protein sequence. SIFT predictions were performed on the identified mutation. SIFT predicts whether an amino acid substitution affects protein function based on sequence homology and the physical properties of amino acids (reviewed in Henikoff and Comai, Annu Rev Plant Biol., 54:375-401, 2003). SIFT predictions with MC<3.25 are considered confident. Changes within a SIFT score<0.05 are predicted to be damaging to the protein.

As shown in TABLE 8, the A111D SIFT score (0.03) is <0.05. This mutation is predicted to damage the SNAP protein. The A111D mutation identified had MC value (3.00)<3.25, thus the SIFT prediction of the A111D mutant can be considered confident.

TABLE 8 SIFT and MC prediction of TILLING-identified soybean mutant. Effect Mutant line no. Nucleotide Amino acid SIFT (MC) F1292 C322A A111D 0.03 (3.00)

SCN susceptibility of Forrest SNAP Type III mutant A111D, as compared to SCN-resistant wild-type Forrest, indicates functionality of SNAP A111D mutation in soybean resistance to SCN (see e.g., FIG. 5).

SEQUENCES >Gm18:1625005..1645004 SEQ ID NO: 1 AAGGAGAAGCAACAAATCAAAGGATGTTTACTTCTGATTTTGGAATGCATCCAAACACTC CTCACGGGGAATTTTTTAGGCTCTCTACTCCCGCGCGATCTCTCTTGTATCACCAGCAAT GAAACTTTGTCATATTTTTCCTTCTCTCTCCACACGAGCCATCTCCTATCCTTCTTTTTC ATATTAGACATTAATTCAAACCCTCTTTCATTTAACTTTCCCTCATGAAAGTGAAATTTA AATGATAATATTGGATTCTGATTGTGCAAATTAAATACAAGTAAAGGCTTAAATAAATAA ATAAATAAATATATATATATATATATTCCAATAAATTAATACGTAATTTTATGATTTTGG TCTTAATAATTTTTTTTAATATAATTTTTCTGTGCCTTTTCTAATAAGGATGATGTCAAG TTAAAATCATGATGATGTCATTAAGTATCGCATCATCAAATGATGAAGTCATCCATTATC ATATTAATAAAAAAAATGATATCATTAAATAGGTCGCCTACATCATCAAATGATATCATT AAGGATTAAAAATAAAAGTTATATATATGCAAGAATAAAAAGTAAAAAAAATGACATTTG TAAGAACAAAAAAAATATTTAAACATTAAATTAAATGTTTACTTCTATATACTCCCTTAT CACACTTATCACTCTCAAGTTTGTTTATACAAATTCTATTTATCAAATATTATCACCAAA CTTTCTCATACTAAAAACTTTTAGTCATGAATGTTGAATTGAGATACCATCTTTCATTTG AATCAGGGACCAAAATAGTAAAAAAATATTCATTTACTAAAATTCAAAATAATATAATTA TATAATTTTTTTGGTGAAAACATAATTATATAAATAAATAAAAGAAAGTAATGAAAAATA TAAACACATATTAAATTAATCTAACAAATAAAAGGTATTTCAATTAGTTGACAATAAAAA ATATACATTATTAACAAGATTATGACTTAAATTGTCTATCCACAATTGCCAATCAAAATA CATCACTAAATATAATTATTATAATTATTGATTAAAAAAAAGCTATCAATCCATATTTTG TAGAATAATACCTTAAAACAAGCATAAACATAAATTCTTAGAACTTAACAAAATACAATT ATTATTATATTTAAATATATATTAATATAAAGTTCAATTTTGATCCTTATAGTTATATAA ATTTTATCTTTTAGTTTCTATACTTAAAAATCATCTCTTTTAATCCTATGCATAATATTT TTAATCTCTTTTAGTTCTTATTATGAGTTTAAACGGTATGGATTAAAAGAAATAAAAATG ATACAAATTAAAAAAGATTAAAAATAATATGTGTAAAGATTAAAATACACAAATTTAAGT ATAAGAATTAGGATAAAAATTATATAATTATAAAAATTAAATGAGTAATTAAGCATTAAT ATAATACTATTATATGAAAAGTTTTGTTTCTAAGAAGAGTCTCGTCCACTTTGCTTTTTA CAATCACATGTTAAAAAAATTATATCATATTAAGGGATGGCCAATTATCTATGCCTATAT AGTTTATATGTTTTGAAAAAATTTGGGGCGTCGTGACTCCCCCGCCCTCAAATAGCTCCG TCAATGACCATTCACATCTTATAGTTCAAGTATCATTGTTACAAGTACACATATTTATTA CAATATATATACCTAAAGTAAGGTTGTTTTTGCTAACTTAAACATCAAAGTGATTTTGTG GATAACCCACTGCCACTCAAGGAGTTCGGTGTCGCAGAGTTCAGGCATCTAAAATCTAGA CCATCGCTTGTCGCACTTTGAACCAAACATGAAGGATGGAGCTTTGAGAAGGAGGGGTGG AGAAGAAAATGGGGTGCAAATATATATAGCGATGAGGAGTATTTTAATATTTAATTGAAT GCTTAAATATATTTTTATCTCCTAAATATGTCATTTATTACTTTTAATTCCAGTAAAAAA ATATATTTTAATCCTTGTAAATTTGTTACTATTACATTTTGTGTTTAATAACATCATCAT TTGATGATGTAACATTTTAATTATACAATCATTTGATGACATAATTTTATCTTTATATTA TATTTGATTACATTATCATTTAGTAATGTGGTGCTTGATGATATTATTATGATGCCAATA TAATATCATCACTATCGGTTCTGATGGTTTTTTTTTTAACAGCGAGAGAAACTAATTAAA >Gm18:1625005..1645004 SEQ ID NO: 2 AGACACATAAAAAAGATAGAAAGTAATATATATATATATATATATATATATATATATATA TATATATGTGTGTGTGTGTGTGTGAGTGTGTGTGTGTATACGGGTTTACAGGGTATGTAC AACATGGTTATATATCATATAGTATCTAACTAACTATATATGAAAATAATTTCATAAATT GTCATGAGTGGTTAGTATAATTTATTGTAAAAATTATTGAGATATGTATGTTTTTTAACT GCATTGAGATATAGTTTTTTTGACTCATAATATATATTTTATCTGCACTCATAATATATA GTATCAAGATTTTTTTTTTTGTCAGCGTCATATGTGATGTTAAATGTGATACTTTATATT GATGAAAAATGCTAATAAGTTTATTTAGCATATTTTTTAAGGAATTAAAAATAATTTTTT TATTCGAATGCATAAAATTGTGTTGTTTATAATTTTTTTTTATAATTTCCTAAATATTTA CTTTTTTTAGTTTTTTAACCCATGAGCACTGGTTAACAAAACCATATATCAATTAGGGGT TATAAATTTTTTAGGCATGATAATTAATGATAATAAAGTTTTAAACTTATGATTTATAAT GCAGTGCAAAAGTCAATTTTCCAAGACATGTTAGGCAAAACTTGGATTTGAATTCTCTGC CATCGGAGAAATCAGGCATTCACGTAGTCAGATGATTAAAAGCGTCGACTAGTGAAGATC TAACAATTCATAAGTCATTTAATAACTTCAACTTTAAAAATATCTAAAAAATACTAACCA TATGATGAAATCAAACGGTCCATAATTATGACTGTGTGAATGCAGAGAATCCAAATCCGT AAAACTTCTGAAAAATGCATATCGAGCGTAAAATTTTATCGAGAATGAGCACGTACTCAG AAGCATCTAGTATGAATTGAATTATACATATAGATTCAGATGTCTACATGCACACACATA TATAATGAGACGGATCTTAGATCATATCATACGTATTTGGTATTTAGAGCTGTTACTTCC TTGTGTTGCTTCCAGTATTGTCACCATTCCACAAGGACAAATCAGGTACCCACCTCTATT CACATCTGTGTCCATTAATCATCACAACAAAACAATATCCTCCATACATATATCCTGCTC CTACCTTGTCTATTGCCTTATATGCTCTGAACTCTCCTAGAGTACTCTTTTAGATCCCAT AATGTTCACCATTCATCAAGGTTTTCCTAATTACTTTAATTAAGTGTTGGAAATGTTGTA TTTTGAGTTGTTGGCCATATTTTGATGAGTTCTCAAAAGACCAGTTTACTTAGAAATTAT TAATTTTTTTTAAGTTAGTATGTTCGCTATATGGCCAAGACTAGGGGAAAAAAACATAAC ATGCATGTAACCCTAGGTCATAATAAAGAATTAAAACCAAGTTATTTTATTAAATAGTAT AAGGGCTATCAATAAATAATTTTTACAAAAATTAAAATTAAAATATTGATATTTTTACAA AAGTTCAACTTCTATGTTACTAATTAATAATATTCTGCAAATAGAAAATCATGTTAAATT TTTTTTTTTAAGATAATTATTATAAAAGTTGATAAAATTATGCATGCATGATTATTTTAA TGACTATTTGAAAGTGTAAGAATTATTTACATTATCTATATATATAATCGTTATTTTCTG AGTTGAATGAACGTGTCCGAGTCATTATCCATATATTTGTATTTTTTTTTTTGAGTATTT TTTTGTTGTTGAATAGTCCATATATTTGATGGTATCAGGAAAATGTGGAAAAGATACAAA ATCATACAGGTAAGCAATTTGTCTTGAGTCGTCATGAGTCTGTTAAAAGTCTATCGATCA GCAACCTTATGTAATATATACACTTTGGGTGCGATCATTTCAATCAAAATCACATTCCCT AGCTTTGGCTCAGAAATCAATAATTGAGCAAGTAATTTTGTGGATACATAAAATAAAAAG AGTTGGCCTGTATGGTGAGGTTCACAGGTAATTACACATTATTCCAGTTTATTTTAGATC GAAACCAGATATTAATGATCACAAAAAAAGATGGATCGAAGAAAATAAAATATTGCTGGC CCTCCCTTATGCTATTTCCTATTTCTTCTTATGCTTTGAACCTATTAATAGCACATTTTA GCCTGAGCAAGGAGTCCGAATGTCCTTTTTCATGGACTTGGCAGTTGCAATAAAGTTGGA GAAGCTGATAAGATTCTGTTTCAGATTTGATTATTCTTACTCACATGTTCCACGATTGAC GAGAATATATATATATATATATATATATATATATATATATATATATATATATATATTGGT TTTTAAGTGTGTCTCTATCCTCCCGTTTAATTTGAATTTGATGACTTTTTGGTAGGGTTA AAGACTAAAGCTAAAACTCCTCTAAATAGTAACTTGTTCTTAAAAAAAATGATTTTTTTT TATCAGAGTTAACCACACATACTTAATTTAAAAGATTTGAACTCCTTTAACTTGAATCAA AAGATGCTAGCTTTTTATTAATGAATATGATGATATGTACGAGGTAATGTTTTAGTATGA TTATTGGAAATTTGGCATCTTGCTAGCATGTAGGTGATACTCTTATTTCTCATGTTAAAT AGTTAAATTCATTTTAAAAGTATAGAATACCAATAAATTGATTCTTAAAAAATAAAAATT TAAATTTAATTATTGAATATGTAAAAAAATATGATAAATTAGTCTTGCAAATAATTTAAT GGCAAGTTAGTTCTTGAAAATATAATTTATTATCAAATTAATCATTAAAAATTATAATTT AATGACAAAATAGTTTCACAAATTTAATGACAAGAATAATTTATTGCATTTTTTACCTAT TTAGAAATTAAATTTATGTTTTTCATTTTTTAAGGACCAATTTATCATCGTATCATACTT TCGATAACAAATTTGACTATTTATATTTCTTATTAACATGCTCAATGATGATCTATTTTT CTGTCTATATAGATAGATCTATGTTCTATAATTTACAATTGAATTATATACAAATATCAT TATATAACAACATATATTAAAAAAAAACTTGTATTTTTTTTATTTTAGAGAGTTTTTGTA TCTTTTTATCTTCCTGTTGGATGAATAATTTCCATGTACATATATACTAGTGCTTTGTAT CATGGAACTTTTACATTTTTTTTTTCCTGGAAACAAGCCATGTTCATAGGCTTAAAAATA ATTAAAGTGACTTTTATCTTTTTCAACAAAGTCTTCTTCATACGCATAACCAACAAATAC ATATTTAAGAAATTCAATCATCTATCAACTGTGTTAAATCTTGTTGATATCTCTATTACA ACACTTTTTATTAATCCGTGTATTAAACTTGAGATGTGGACTTAATTTTAACAATTAATT AGAAAGCTATGCTAACTAAATGAGAGTAATTACAAGTAATAGAACCAAAATACAATAAAA ACGTTCCAGAAATTAATGTTCAACTAGTATATTTTTTATGAAAAATAAACAGAAAAGTTT TTAAAAAAATAAAGGGTTATAAATCACCTTGGTTGACACCCAATGAGATAATGGGCTAAA ATTACTCATTCATTTCAAGCCCAGTAAGTCTGGGCCTAGCATTAGCTTCAAGTAGTTCGG ATTCACCCGACCCAGATAAGACATTCGGGTTCGGATCCTGTAGTGTCATCACTCGTTAGA TTTCGATGAAGAATAGTTAGAGAGTGCTGTGAGCTTCAGCAAAATGCGCGCCCTAGCAGC TCAGTTCTCTAATGTAATGTTCTCTTTAATCCTTGTCTCTGCCTCTGTTCAATTTTAACC CTTTATAATTTAAAGAAGAAAATAAAGTAATCATTTTCAAATTCAATTCATATTTGAAAT CGATGTTGATCTTAAGTAAGTACCCATTTGCGGATTCATTTTATTCTTAACTTTTTTCAT TTTTTTAACCCTTTTTGCAGTATTTATGCAGAAGAAAAGTTGGGGTCAATCTGCGATCTC GTAATTTTTCATCATATAACAGTAAAGGTATCACCTTGTTTTTTTCTATCGGTACTTTTG AAAGAAAGAAATAATTATAAGAAATTATGACTAATTTTGTCAGGCTATGGAATCATTCTG TTGAGAACGAGAAAAATGAAATAGGTACTTGTATTGAACGTTCTAGTAGTAAATGATAGG TACATATGTTGATTTTGGGGATTTCTGGGATTAGATCATCAAGCCAAAATCTTGCACTGT AAAATACTAAAATACAAAGAAAATGGTTTTCTTTTTTCGTTCAATTTTTGGTTTCATTGT TTGAGCTGCAATTATGTTGCGTGACTTCACTTGTAACTCTTTTGATTTCCAGATGAGCTA ACCATCGAGGAAGAAGCTGAGAGAAAAGTTGGATGGCTATTGAAGACGATATTTTTTGTC ACTGCAGGGGTAGCAGGATACCATTTCTTTCCTTATATGGGTATATCAGCAAAATCCCTG CAACAATTTTTAACTTGCAAAGCCTTCATTTGCTCTGGGTAGTTCAAGCTTCACAATGCT GTTAAAATTCATATTTTCAGGAGAGAATTTGATGCAACAGTCTGTTTCGCTTTTGCGTGT CAAGGATCCCTTGTTCAAAAGGATGGGAGCTTCTAGATTGGCTCGTTTTGCAGTAGATGG TAAGTTTTACTATCTGTATCTTTGTGTCACTAATTGCTTGCTGTTGTTGTTTTATGACCC ATATTTCTTTGGCACATGACATATATATTGAATTGTTTATTAATTGTTAGTCATTTGCAT ATTAGGGCAGTTGTTTTCTAGAACAGATTCCTATTCTTGCAACAAGCATATTTTCATTAT CCTTGTGCTTTACACTAGTGACATTTAGTCATTTAGTATTAATCTCAGCTTATTCTTGAA ATGACAATTTTGGTTGAAGGGGAGAGTTGATGGGAGATTTTGAACTTGGATAAAAGAGTC ATAGATTGAAATTTTTCTCTTGAACTGATAATCAAATAGTTATTGAGATTTTTAATTGAG CTGCATTTGTTAAGAAGTCACGGCTAAAAGAGTTACCTAGTTGTCAGTTATACTATTTTC ATGACTAAGCAGCAAGCACAGATATTGCAGTGATACACAACCGAGAGCATATTCTCCAAA AGGCAAATTGCCTTGATGCAATTTGCTAGTTTGTACACTGATAGAATTGCTTATTTATCA ATAGTGTTCCAATGTATAGGTATCTCATGGCACTGATTAAGGATAAAACATGGTGATTTA TTCCTTTCTAAAATCTTTTGTCCCTGCACAAGTTGTTTATATTAAACATTGTTTACCTTA CTTTTGCTCACAAGATGAAAGAAGGAAGAAGATAGTTGAGATGGGTGGAGCTCAAGAACT CTTAAATATGTTAAGCACTGCTAAAGACGACCGTACACGGAAAGAAGCATTGCATGCTCT TGATGCACTGTCACAATCAGGTGAAATCATAATTTTAATATTTTTTTAAATAGTTATTAT CATGCTGGTGGAGAGGTAGATTATTGTGATCAATTAGTACCTTTGTGGTTCTAAATAGTA AACCAGAAATGCCCAACCACTTATGGAATTTGTTAATTTATTTGTATAATATTGAGCTGG AAATCAATTTTATGAGCAAAGTGTGATAAGAAGCATATGAAACTTTTATATGTTTCCAGT TTCTGTTATCCTTATTCAATAGATATGGGCTTGTAAAGATGAAAATGAACATAAATTGTT TTGTGCATTAATTTTGGGACATATTATACGTGCACAAGCTTATGTGTAACAATATCTATA CCTGCTACTTTCCCTGTCAACATATTGATTTTTAAGAATCCAGTTCAAGTAATATTTATG AGGTTGAAAGATATGCAACAGTACAACCAAATTAGTAGTGCTAGCTAGTACTAGCCTTTT CTGTTCTCTTTCGATTGAGATAAGCTACTTCAATGTTATAGATGAAGCTCTTGCATCCTT GCATCATGCTGGGGCCATTTCAGTAATTAGGTCTGCACCAAATTCACTTGAGGATGCAGA AGTTGAGGGATTCAAGTTGAGCTTGATGAAAAGATTTCAAGATCTCAGATATGATGTGCC ATCATGACTTGAGGTGCATGCCTCCTTTTGCTTTATGTTTTTGGTTGGTTGGAGCATGAA ATAACATGATATGAGAAATTAAGCTGGCAACCAAAGCTTTTGTGGGGAAGAGTACTTGAA ATTACTGTGTATCATTTGACCAAATCTAATGGAAGATTATAGTTCTATTGTCATTTTAGT TTTTTTCACTTGTCAAATGCGATTTGTCGCTCATTGTTCTGTCAATCATAATAAAATGGA AAAGATTTATGTGCATGTCAATTTTTATTTTTTGAAATATGTGTTTAGAAGATAAAAGAT TACAACAAACTAGTATTGAAGTTGTAAGTGTTTAGATACTGTAATTGTATATTTGGTTAA CACTACTAGATTAAATTTAAGCCTCAACTTTCAAATGTGATTGATCATATAATGTCATAA AATGTGTGTAATTATAGGTTGATGCTTTAATTGTTATTTACATATGCCTCAACTCTCAAC CGTATGTCATCATCAGGCTTCTTGTTTAGGTTCAGCTGGCGCCTTGCTCGTTCTATTTTG TTCGTATTCCTTTGTTCATTCGATGTTTTTTTAGGAAAATATGTTCGTTGAAAGAAAAAA TCAGTCAACAGAAGATACATGCCCTTACTTTTCTCTACTCCACGTCTCCACCTACCCTAA CCCTTGGAGTTACTTTTCAATTGAGCACGTTAACAAGCCTAACTAAACGTGGTTCTGTGG AGATAATATACTAAAAAAATATTATTTTTTATTTAATTTAATAAGACTTGTGCGCGTAAC TCTTTTCAAAGTGCTAGCTTTCTTTTTTGGTGAATTTTCAAAGTGCTAGGTGAATATGCG TATTTGGAGATAGAAAGCTTTTTTTTTTGGGACACAAAAGCTTGTTTAACATGTGATAAA CTTAAAACTAATAATCATTTTTTTTAATTATCCCTATTCAATGTTTTAGTTTTAAAAATA CCCCATTTGGGAAAATAGCCCATCTGTGGATGTAAAAATTACTAGAGTACAAGTTAATTA GGGTTAGTTGTTTTTTTTTTCTTTCTTCTTTTCCTACGAGATCAAGAGGAACGGAGCAAA ATCATGTTTTTTCTTCCACCAAAATAATGTAAAATTTTAAAACAAAATACTAAAATAATA GGTTATAATATATTTCTTTTCCTTCATTTTATACTTAGTCTTTTATTTTTTTTAAAAATA TCATTCTAGTTATTTAACTCTCACATTTGAGTTAATGTTATACTTTTGAAACTTCAGTCA TTTTTTATTTATTTTGGATACGATTTTGAGCCTTTTTTCTTTTACTAATTAAAGATATAA AATTTGTCTCATTAAATTAAACTATGAATCCAATTAATGTTCAAACAAAACATAAATCCT TAACATGATGAAAATTGAATGAAAAATGAATTTCATAAGCAATTGGAAAACTGAAAAAAA AAATCTAATAACAATATTTGAAAATCTATTAACAATGAGCAGTAGGCTTCTTTGGAACTT GAAATGAAGAATAAAAGAGTCATTGGAAATAAATCTCAATTAATTGAAAGATATTTTTTA GAAAATGTCATTAAATAGAATAAAAATAATCAAAATTTGCTTATATTTGAATCTAATAAA AAAATTGTTAATTACTCTTTTTGTCTCGAATATAAACAAAAAATACTTTGAGTATTAGTC TCAAATAAAAGTAAAATTTAACTATTGTTACTTTATTTAATGAGATATTCCTAAATTATA TTTTATTTAATTAAAGTTTTATTACTTATTATCTCTCTTTTTTATTTTTGATATGAACTT TCAAGAAAGTGTAGTTTAGAAGAAGAATTTTTTTTAATAAGAAAGGTGTAATTAAATAAT CTAATTATCTAACTACTAACTTTTTGAATAAACCGTAAGTTAATTTCTTTTATATAGAAA GAAGGGATTAAATTAATTTTAAAAGAGTTCCTCTTCATTTTAATCATTAATTTTTTTGAA ATTCAATAATCAATACCTACTCATTACAATAATAAAATACAAAAACTTCTTCACGAAATA >Gm18:1625005..1645004 SEQ ID NO: 3 AATTGATTCCTTGTGTTATATATATATATATATATATATATATATATATATATATATATA TATATATATATATATATATATATATATATTCAGTATATAATTTAGTGTAGCTGGACACAT ATTAGTGCCCCGTGGCCGTGTGTTGTGCTTTTTTGTTGGGCGAAACAACGGCCAAAGCGA CGAATCACTTCCTTACGTGACACACCTCTGTCTAATAGACGATAGGCCAAAGTCACAAAT ACTTTTTGATTGAGTATTTTTTAATGCCACATATCATATCTGTCAGCGTCACATGTTCAA ATAAATCCCTAGTAAAAACGCCAGAAAACAAATGCATGAGCAATTTTTGGACTTTGGACT AGTTACAATTTTTCAACGTCACATCTTTAAATGATTTCGTCTCTATTTAGTAGTTGTTTT TAATGCGGCCATGCCCACTTTCTCGTTACAAAGCATGCATTTTATTATTTGAACGAAAAT ATTTAATATTGTGTATTACTCGTTTTACACAATTGCTTTTATTCTTTTTTTTTTATTTTT TAAATACAGTCATCTTTAAAAACAAAATCTTCGATCATTCCATTTCATTGTTCACAAAAC ATTATCCTATCACATGCACCCTATGTAATATAATACACGGTTGTGGATAAAATAATTCTG CACCTGCCCAACTTTTGTATTGATATCATTTTTTTATTTCCTCTATATTTTCCATATTTA TATATTAATTCTATCACTTTCCTGCACCCCAATAAGTCATGCTCCAAATATTATTGTTTT GGATAAGTATATTGCACAATCTATTCTTGGCTTCTTCATGACCATGACACGGCAATGGAG ACGAACGATAATGAAAGGCGCGTAAATCATTTGAAAGGTTGAATTATGTACCAAATGCTA TTATATTAGCATTTCATACGCCATTCTAACAATAATGAGAACGAGTTGCCCCACAATTGA TCAATATTTGTATCCTTGCACGGCACAAACTTGTAAGATATGGCCCCAACTTCGTCACCC CATCAAGTTGATTTCATTTCTTTAATATTTGGTATTTTACATAGAAATGCTGCCACAAGA CATGAATTCTACAATAAACAACAAGGGATCCAAAATACAAAAGTAACACAATCGCCACAG CAACCAAATGCCTCTAGAATCTCCCCAGCCATACCCTCTCTCCCCAAATAAAATTTTCTA TACATATAAAAAAAACAATAGAAGGGGAGAAATGAGAGAACCAGCTTGTATTTATGACTT GCTACTAAAAGCATTATATATGTTGGTGGAAATGGCAAGCACACTTGTAACCACAGCTAG TATAATCATTATCAGTGCAATAATTTTGTCTCTTCTCGTTGATATACCTTTAACATCCCT GTCATCAGAAACAATGAAAATTAAGAGACTAATTTATTTATTTATTCAATTCAATTTTCA ACTTGAGCTCTACTTTATCAGAAGTCAATAAGTCTATGCTAGATTACCTTAAAACAATAG AGCCGGGGAAAATGAAGGCAAGGCACACTGCGGATGAGGATCCCAGGAACTGAAAGAAGT ACCAAATATCTGGGATTGCTATAGCTGCAAGGTAGGAGAATACAAGCAGCACCAGAGTGA GGATCATAAATCTTTTGTTGTCTGTGGCTAGCATAGGCTTCTTAGGGAAGAGAACTTCAT CTATGTTGGTTCTCAAAGAGAAGTTCAAGAGAGGAAACACCAGCATGATGTGGAGGGCAT AGCTTACACGGACCAAACTATTGAGCAAGGAACCAACTGCTGAACCAGCATTCTGGTCAA AATTGATGAGAATGTCTGACTGGGTTGAATCCCCAAATAACATGTACCCAAATAAGCCTA TTGCAAGGTAGATCACAGCACAAAGCAATAATGCTAATCGAACTGCTGTTGTCATTTGGG ATGCCTTGGCAAGCTCAAACCCAATGGGGTGCACTGAAAGTAAAATCAACCATCAGCACA GAAATTTTCTAGGCATACCCTTAAAAAAAATGAGCAAATCCATTAGGCCAATTACCATTA AAGTGAAATGTGAAGGCTGTGACAACAACAGGAACTGCAGTGAACAGATCAAAGAATGAG GTTTGGTAGTCTAGCCGAGGAAACAATCTAGGAGTTTGTGTTTTTCCTTGCACCAGAGCT GTGATAGCCAACCCACAACATATGCCAACAAATGCCACTGCAAGAAGAGTTGACACTGCA GAGCTGTACTTCAAGGACTCTACCAATTCCAAAACACCAAGAGGAAGAACCCATGTTAGT TACTGAAACTCTTCAATTCCAAATACACAAGAAAGCATGTTATATAACAAAATATTCAAT TGTAACACTCACCTACACGTTTGTACAATACCAATGGAAGCATAACAAAGACCAAGGTGA AAAGCAAAGCAAATTCCCGGGAATTCCACCAGTGAATTCCAAACCACTGTTGCAAAATGC CCAAATGCACTTCCCCTCCATTTTGCTTTCCAGATAGCACATCTCCTGTTGTTCAACAAA AGGTAATTATTAAAATACATCTATTTTTTTCTTTCATCTTCTTTCTACTACATTTTCTTC TTATATTTCTCTTTATTTCCTGTCATTTCACTTTTTTTTTTCCTGTCTTTCTTCTACCCA TTACATAGACCAAAAATTGAGGTGTGCATTGCGGAAACAGATTCCCCACATTCACTTTTT TTTTCATGAATCAGAACTATTAGATTGAATATCGAAGGTTATATTTGGAATACATATTTT AAGAGTATGTTTGGATACAAAGTTAGAAGTGCATTTGACAACTTTTGGGAGCTCCTCTAA TGGAAAAAGATTAATATTTTTAGTAGAAAACTCTCAAAAACACTTCTAGTTTGTATCGAA ACAGGCCTAAGTTAGATTCATTGAGATAAGTTATGAGTAATACAATCTGTATTTAACAGT GAATTCCCAGCGGTAGGAAATCCCGATCCTTACATTTCCATTATCTTGTTAAATTAATTA CCATACACAAACATTAAAGAAAACGACTTACTACATATATTTTTTCAAAAAATGAACCTT TCTATTTATAGTAAAAAAAATAAAAAACAAATTTCACTTATTATTAGCAACAATTTCACC AATCAAAATGAATCTGACTGAAAACCCGGCAAAACTCAGAACAAGCATACCAAACCAAAA ACATGAAAAAATCTACATTTTTTTTTCCTTTTTTACGAATTCAGTAGAAAGGAAATTAAA AAAAAAGGGAAAAGTTCCGTTACGTTACCGATGATGATAAGGTAGAGAATTAAACCCCCA ACGTTGGTGATGATGACGCAAACTTGCGCGGCTAATGCTCCACCCGATCCGAACGCCTCC CTCATGACGCCAGCGTACGTCGTCGTTTCGCCGGAGTGCGTGAACCGCATCAGGAAGTCC ACGGACAGTTCCGCCAGCACGGCCACCACGAGAATCATCGCGAAAGCGGGAACTACGCCG AGAACCTTCATGATCGCCGGAATCGACATGATTCCGGCGCCGACTATGCTGGTGGCCACG TTGAACACCGCGCCGGGGACGGAAGCCGGCGGCGGCGTTCCTTTGGAATCCCCCAGGAGG GGGACGCTGACTCCGGCGGCCGGAGACATGCCGGAGGCAAAATTGTGAGGATCGGAGAAA GTGCGGTGGCGGTGTGCGGTGCCTGGCAGCCTACTATCTTTGAATTGAATGGTTTTTGTT TTGTTGTCTCTCACGAAAATTTCACTTCCTCTCTCTTTATAAATGATACAAGTGGATTTG GGAAGTTAAGAAAACAAAAAATGAAGTTATAATAAGTAAGATTTTATTTTATAAGTTTTG TAGGATGAAAAGAAATATAGTTGAATGAGGAAATTTCATTGAAAATAGTTAGCTAGATTT TATAATAGAGATTAAACAATAATAAAATCTGCAGATACTTCAACATGAGTATGATAATAA TAATAATAAAAAATTGTTGTTTTCTATTTTTACTCCAACATGGACTGAAATTCATATGAA TTTTTTTGAATAGTCTATTTTTTTTTATTTAATTTAATATTCATATCAAAGTTATTTCAT ACTGAAAAAAATATTAAATACTAGCATTCTATTATTACCATTTGGAGGAATGATTGAAAG AGTGTTAAAGTGCACCTTTTCAGTCAACAGTTAAAAATAAGGCGTTTAATTCAATTCAAT ATTACAAAGTTAAGTTGGCTGTATAATAATAACAGTGGTAGTAAGTAGTAGAGTGAAAGA AAAATTTTTTTGGTCAAAATATTTAAATCAAGACTAGAAGATATGCAAATCAGAGATTAC ATTGGATGATACGGTCGACCAATAAAAAATAAAAGAAAAAACATAAATTGGGATGTTCAA ATACTAATAATAATAACTCTAAACAAACATTAACACGTGAGTTTTCTTTCCCACGTTGTA ATCATTTTGAATTTTTAAAATGTTATGACACAAATAATAAGTTAATAATAATTATAATTT AACATTTGAATTGATAAAAGTGTTTAGTTTTATTGTAGATTAAACTAATCTTTCTTCGAG TAAAAATAACATTAAATTCCTACACAACAGGTTTATCAGTTTATAGAGTAATAACACTCT TATTCTTAATCGTTTTCTTTTCTGGAAGAAAAAATAAATCTTAGTCTTGTTATTTTTTTG AGAATGTAAAATATACCTTAAAAAATTCCCTTAAAGTTTGTATAATTTTTTGGTATGTAA ATATATTTATAAATAAAAAAATGTTTGCGAAAAGTAATATTTACATAACAAACACTATTT ACAGAACATTGATGAAATTATTTTTAGATATATAATTATTAATACGAATATATGAATATG TTATTAAAGTAATCAATAGTTATGTTAAAACTGATCTGTTGACTAGACAGTTTGTCAATT TATTTTTTATTCACTTAATTGCTATTTTTTTCTAGGTTTGTTCTTTCGTTAAAAAACCTT GCATTGGAGGAAGGCCAATGCTAGTTATAAAAATATAAACCATGATTTGAATATAAAATT ATTTTTAGTCGAAAAACAATGAATTATGTTGCAAGTATCACTATTGAAAAAATGCCAACG GAGCCCAAGAAGGTGAGGCCCAAACTGAAAGCGTGAAGCGGCCCAAGACTGAGTGAGGAA ATAAATAATTATCCAGAAAATCGGAAATGGACAATCCTTCTTGTTACGCAATTCTGAATT TGCGGGTTTTGGATTTGGACTTGGTCGTCAACACAGTCTAATTAATATCTTTTTGCTCCT TCGCTTATGAATCTTCTTCTTCTTCTTCTTGTTCCTGCAACGCACTGAATTCGATCAATC AATCCATCTTCAATTGCTTTGTTTCGATCGGAGGAAAATGGCCGATCAGTTATCGAAGGG AGAGGAATTCGAGAAAAAGGCTGAGAAGAAGCTCAGCGGTTGGGGCTTGTTTGGCTCCAA GTATGAAGATGCCGCCGATCTCTTCGATAAAGCCGCCAATTGCTTCAAGCTCGCCAAATC ATGTTTTTCCTCTTTCTCTCTACTTTTTTTAAATTCCATTTCGTGTCTCCTCAAAATGCT GATTTAGTGTCATAAATCATAATTATTATTCTCTTCTATTGTTGTTATTTTATTGTTATT ACTTCAATCGACGAGTGTGTTGAGTTTTGAGGTGTCCGATTTCCCGATTAATTGAAGTAT AGTTTTAATCTGATTTTACTGGAAAATATTTTTTGCCTGATTTTTTTTTTTTGGAACAAT TACTAGCATATAAATTAGAATTGTGGATGAAGTACGACAATCAACTCTGTGTTGTTTGTG ACTGCGCTCACTTTCAATTTGACGACTAATCTCTTTATTTTGTTGAAAGTGACGAACTTT GAAATTGATGTTGGAATAGTTCTGTTTATTGTTCTTGATTTGATCTATGTGGCATTTTAG GGGACAAGGCTGGAGCGACATACCTGAAGTTGGCAAGTTGTCATTTGAAGGTAACATTCA TCAGACTTGGGGTTTTGGAGTGGGCTGAATCTCTTTTGCATCCTTTAGTTCTCTATTAAG CCTGCATGACATTGTTGTGTTCTGTTTCCATTTAGTTGGAAAGCAAGCATGAAGCTGCAC AGGCCCATGTCGATGCTGCACATTGCTACAAAAAGACTAATATAAACGGTATGCATGTGT CTCAGTTGTTACCACTACATGCACTACAATACTTTCTCATTTATGATTTGTGCTTTAAAT GCTGCTCTTGCTTCCATGCAGCAAGGCCAATTCCTTTTAGCCTCAATGTTTCTCTGTATA ACTTTAATGTAAATCATATAAAACAATTGCTACCTTTTTGCATGAACAAATTATATAAAG CAAATCTCTTTGTTTAATCTTTACATATGTGTAAATCAAATACTGGGCTTCATATCGATA AGGTCTAAGTAGGGGTTCAGTCTTTTATTTGGATTAGTTTAAGTCAGAAATTGAAGTTAA TTTGTGCTTGCATAAGTTGCTTCCATCTGATTGCTTTCTTTTTATGGCTGTCTGTATGTC ATAGCCTTATTTTGATTTGTTATTTGCTGACTATTATTAGATTGGAACTCATGATCATAT CCCTAAGCAGGAGCAAATTATTTTGCTGTCTTGCTTGTCTTAGTATGTCCCACTTGCATT AGGAAGAACTAAGACAATTAAAGTTACCTTTTCTTTCTTTGAATACAGAGTCTGTATCTT GCTTAGACCGAGCTGTAAATCTTTTCTGTGACATTGGAAGACTCTCTATGGCTGCTAGAT ATTTAAAGGTATATTATGTTTATGATATTGATATCTCTTCTCCTGGGTATGATTTTTAAT TTATTCTCTTGTCCATATCCCAGATTTTAGATATTGATCCTGCAATAAAATGCGTTGAAG TATACTAAGTTATCTGAATCCCCATTAACATGTTTTAACTGGGTTCACTATTTTATACAC AGGAAATTGCTGAATTGTACGAGGGTGAACAGAATATTGAGCAGGCTCTTGTTTACTATG AAAAATCAGCTGATTTTTTTCAAAATGAAGAAGTGACAACTTCTGCGAACCAATGCAAAC AAAAAGTTGCCCAGTTTGCTGCTCAGCTAGAACAGTAAGATATTGTCCTTTCTGCATATA TTATCTCTTTTATTATGCTGATGAATTGATCAATATTTCTTCAACTTGGGTTTATTCTTT AATTGGTTAGTAATTTCTTCTGAGAACTTTCTTTCTGGCCTTTATTTTGTTCAGTACCCT TTCTCTAACCCACTCTCCTCAGGTTAACATTAGCTTAGGTCAGTGTAGGTTGTTTGACAC TGAGTTTTTATTGGTATGGATGTATGGTCTATTATGATCTCAATGGAAATCTAGCATATT TTTTTTCCACAATCCATAATATGATGACTTGTGTACATGGTGTGAATAAAAGTCAGTCCA TTGCTGCATTTGGTATTGGTTACGTGTTACTGTACTTTCTGCATATATTATCTCTTTTAT CATGTCGATGATTTGATTAATATTTCTTCAATTTGGATTTATTCTTTAATTGGTTAGTAA TTTCTTCTGTGAACTTCTAGTTAGAGCATGAACTGCTAAAGAAATCCAAAACTTTATTTT TTACATGGAAGGAACTTTATCAGAGTTTTATTTATTTATTTATTTTTATGTTAAATTGAA CTTTAACTGTTTCTATGTTATGATAACTCTTCTTCAGATATCAGAAGTCGATTGACATTT ATGAAGAGATAGCTCGCCAATCCCTCAACAATAATTTGCTGAAGTATGGAGTTAAAGGAC ACCTTCTTAATGCTGGCATCTGCCAACTCTGTAAAGAGGACGTTGTTGCTATAACCAATG CATTAGAACGATATCAGGTCTAAGTTTTTTCAATAGTTCACTTCTGGAGACTGGACAGCT TATTTGTTGCTAAATTATTCAGATATGTTTTTATTTTGCAGGAACTGGATCCAACATTTT CAGGAACACGTGAATATAGATTGTTGGCGGTAGGTCACTGGTTTTGAAATTTCGTTATGA ATTTTTTATGACCAAGTAAATTGGATTAGAATATTTGAACTTCTTTGTAGCTGTCTCCTG GGTCATAATGTTTTATTATATTTTTGTATTTATCATAGCATTGTGATAGCCCTGTTACTA CTTTGTTTGCTGATTTACTCATACATTTGCCAGATGAAACTGACATTTTTTTTTAATCCT GGTGGATAGGACATTGCTGCTGCAATTGATGAAGAAGATGTTGCAAAGTTTACTGATGTT GTCAAGGAATTTGATAGTATGACCCCTCTGGTAAGCTCCAAAAGTTGTTAAATAGGATAA CTTCTAGTGGTGTTTAACAAAAAAAAAAATTCCACTTGTATTTTTTATCCACATTTTATA ACAGAATAATCATAACCTTTCACAACTTAATTCTCAATTTTCACAGTAATTAAATGTGTA ATTTTAAAAAAATATTTTCCTTAACTTAAACCTGATTGAAATTTCCCCCTGAAATTTAAG TTCTATTTGATTACCTAGAGTGTAATTTCCGTGTTTTGTCACTTAATCACTGTGTAAAGT TAATTTTTTTGCTTACAAAGGTGTCTTGTTTGGAATGCTAAAATAACAAGTACACGTGTC ACCAAATTTAGTAGGATTAACATTTGTTGTTTTTTGCCATAATAAACGGTTGAACTTAAC ATTTGTTGTACGTGTCATCAAATTCTACAAATTGTGAGCTGCTTAGTGGGTTGGACAAAC ATTTTAGCAGGTGGTTTCGATTGCCTGTTGAATACGTGAAATTAAACCAAGGCAAAATTA TAATTTGTTTCTTTTGTCTGTGTTTCACTCATACACATTGAATCTTGATGATACACAGCC TTGTTAATTGTTATCCTTCCAATTTTTTTTAGTGTTTTTGAGCATCTATTCTTGTTGGTC ATGTGTTTTCTTCACTCATGTACCTGGTTCTTTTCCTACAACGATAAATATGTATCCTTT GTTTTTTTTTTCCAACTAAATATGTAATTTCAAATTTCTAATCAATCATTGCTTCCAAAA TACTCTCTCTGTTTCAAAATAAGTATTATCCTATATTGTTTTACAAGACCAAGAAAAGCT AATATATAGATGAAAGAAATTAGTAATTTTACAAAACTAACCTTAGTATTAATATTATAC TGAAAAACTAAATTGACACTTATTAGGGGTGTTAGTGTAAAAAAGCAATTAATATTACAT TGAAAAGCTAACATGATACTTATTTTGGGACAACTTTTTTCTTTCAAATGCAACACTTGT TTTGGAACGGAGGGAATACTAGATATTGTGCTCCCTTGTATGCCCTGGACATAACGTATT TAACTGGTCTGGATGAGTTTATGAATGTCATTAATTTAGGGGGAGTCATTTAGAATAGCT TACCTATAAGTACTTTCTAACTTTTCTCAATTAGTTTCACAGTGCAATTTATTAAAAATG TCTGTATCTAATCAACATTGTCTGTGTGCTTGTGCAGGATTCTTGGAAGACCACACTTCT CTTAAGGGTGAAGGAAAAGCTGAAAGCCAAAGAACTTGAGGAGGATGATCTTACTTGAAT TGTACCTTTAATATTCCTGG Glyma18g02570.1:peptide SEQ ID NO: 4 MRALAAQFSN YLCRRKVGVN LRSRNFSSYN SKDELTIEEE AERKVGWLLK TIFFVTAGVA GYHFFPYMGE NLMQQSVSLL RVKDPLFKRM GASRLARFAV DDERRKKIVE MGGAQELLNM LSTAKDDRTR KEALHALDAL SQSDEALASL HHAGAISVIR SAPNSLEDAE VEGFKLSLMK RFQDLRYDVP S* Glyma18g02580.1:peptide SEQ ID NO: 5 MSPAAGVSVP LLGDSKGTPP PASVPGAVFN VATSIVGAGI MSIPAIMKVL GVVPAFAMIL VVAVLAELSV DFLMRFTHSG ETTTYAGVMR EAFGSGGALA AQVCVIITNV GGLILYLIII GDVLSGKQNG GEVHLGILQQ WFGIHWWNSR EFALLFTLVF VMLPLVLYKR VESLKYSSAV STLLAVAFVG ICCGLAITAL VQGKTQTPRL FPRLDYQTSF FDLFTAVPVV VTAFTFHFNV HPIGFELAKA SQMTTAVRLA LLLCAVIYLA IGLFGYMLFG DSTQSDILIN FDQNAGSAVG SLLNSLVRVS YALHIMLVFP LLNFSLRTNI DEVLFPKKPM LATDNKRFMI LTLVLLVFSY LAAIAIPDIW YFFQFLGSSS AVCLAFIFPG SIVLRDVKGI STRRDKIIAL IMIILAVVTS VLAISTNIYN AFSSKS* Glyma18g02590.1:peptide SEQ ID NO: 6 MADQLSKGEE FEKKAEKKLS GWGLFGSKYE DAADLFDKAA NCFKLAKSWD KAGATYLKLA SCHLKLESKH EAAQAHVDAA HCYKKTNINE SVSCLDRAVN LFCDIGRLSM AARYLKEIAE LYEGEQNIEQ ALVYYEKSAD FFQNEEVTTS ANQCKQKVAQ FAAQLEQYQK SIDIYEEIAR QSLNNNLLKY GVKGHLLNAG ICQLCKEDVV AITNALERYQ ELDPTFSGTR EYRLLADIAA AIDEEDVAKF TDVVKEFDSM TPLDSWKTTL LLRVKEKLKA KELEEDDLT* Forrest SNAP Type III polypeptide mutant A111D: SEQ ID NO: 7 MADQLSKGEE FEKKAEKKLS GWGLFGSKYE DAADLFDKAA NCFKLAKSWD KAGATYLKLA SCHLKLESKH EAAQAHVDAA HCYKKTNINE SVSCLDRAVN LFCDIGRLSM  D ARYLKEIAE LYEGEQNIEQ ALVYYEKSAD FFQNEEVTTS ANQCKQKVAQ FAAQLEQYQK SIDIYEEIAR QSLNNNLLKY GVKGHLLNAG ICQLCKEEVV AITNALERYQ ELDPTFSGTR EYRLLADIAA AIDEEDVAKF TDVVKEFDSM TPLDSWKTTL LLRVKEKLKA KELEEYEVIT 

What is claimed is:
 1. A transgenic soybean resistant to soybean cyst nematode (SCN), or a seed, plant part, or progeny thereof, the soybean plant transformed with an artificial DNA construct comprising, as operably associated components in the 5′ to 3′ direction of transcription: (a) a promoter that functions in a soybean; (b) a transcribable nucleic acid molecule comprising (i) a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 (Glyma18g02570), SEQ ID NO: 2 (Glyma18g02580), and SEQ ID NO: 3 (Glyma18g02590), or a nucleotide sequence at least 95% identical thereto encoding a polypeptide having Glyma18g02570, Glyma18g02580, or Glyma18g02590 activity, respectively; (ii) a nucleotide sequence encoding a polypeptide comprising SEQ ID NO: 4 (Glyma18g02570), SEQ ID NO: 5 (Glyma18g02580), SEQ ID NO: 6 (Glyma18g02590), or SEQ ID NO: 7 (Forrest SNAP A111D mutant), or an amino acid sequence at least 95% identical thereto having Glyma18g02570, Glyma18g02580, Glyma18g02590, or SNAP activity, respectively; (iii) a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, wherein the polynucleotide encodes a polypeptide having Glyma18g02570, Glyma18g02580, or Glyma18g02590 activity, wherein said stringent conditions comprise incubation at 65° C. in a solution comprising 6×SSC (0.9 M sodium chloride and 0.09 M sodium citrate); or (iv) a nucleotide sequence which is the reverse complement of (i), (ii), or (iii); and (c) a transcriptional termination sequence; wherein the transgenic soybean exhibits increased SCN resistance compared to a control not expressing the transcribable nucleic acid molecule.
 2. The transgenic soybean of claim 1, wherein the nucleotide sequence is at least 95% identical to SEQ ID NO: 3 (Glyma18g02590) having one of more mutations selected from the group consisting of C163225G, G174968T, A164972AGGT, C164974A, C163208A, G164965C, G164968C, A164972AGGC, and C164974A; or the encoded polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 6 (Glyma18g02590) having one of more mutations selected from the group consisting of D208E, D286Y, D287E, -288V, L289I, Q203K, E285Q, D286H, D287E, -288A, L289I, and A111D.
 3. The transgenic soybean of claim 1, wherein the encoded polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 6 (Glyma18g02590), a mutation of A111D, and Glyma18g02590 polypeptide activity.
 4. The transgenic soybean of claim 1, wherein the transcribable nucleic acid molecule is expressed in epidermis, vascular tissue, meristem, cambium, cortex, pith, leaf, sheath, root, flower, developing ovule or seed.
 5. The transgenic soybean of claim 1, wherein the promoter comprises an inducible promoter or a tissue-specific promoter.
 6. The transgenic soybean of claim 5, wherein the promoter comprises a nematode-inducible promoter.
 7. The transgenic soybean of claim 1, wherein the promoter is selected from the group consisting of factor EF1α gene promoter; rice tungro bacilliform virus (RTBV) gene promoter; cestrum yellow leaf curling virus (CmYLCV) promoter; tCUP cryptic promoter system; T6P-3 promoter; S-adenosyl-L-methionine synthetase promoter; Raspberry E4 gene promoter; cauliflower mosaic virus 35S promoter; figwort mosaic virus promoter; conditional heat-shock promoter; promoter sub-fragments of sugar beet V-type H+-ATPase subunit c isoform; and beta-tubulin promoter.
 8. The transgenic soybean of claim 1, wherein increased SCN resistance comprises at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1000% decrease in susceptibility to SCN as compared to a non-transformed control.
 9. The transgenic soybean of claim 1, wherein the transcribable nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 (Glyma18g02570), SEQ ID NO: 2 (Glyma18g02580), and SEQ ID NO: 3 (Glyma18g02590).
 10. The transgenic soybean of claim 1, wherein the transcribable nucleic acid molecule comprises a nucleotide sequence at least 95% identical to SEQ ID NO: 1 (Glyma18g02570), SEQ ID NO: 2 (Glyma18g02580), or SEQ ID NO: 3 (Glyma18g02590), and encodes a polypeptide having Glyma18g02570, Glyma18g02580, or Glyma18g02590 activity, respectively.
 11. The transgenic soybean of claim 1, wherein the transcribable nucleic acid molecule encodes a polypeptide comprising SEQ ID NO: 4 (Glyma18g02570), SEQ ID NO: 5 (Glyma18g02580), SEQ ID NO: 6 (Glyma18g02590), or SEQ ID NO: 7 (Forrest SNAP A111D mutant).
 12. The transgenic soybean of claim 1, wherein the transcribable nucleic acid molecule encodes a polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO: 4 (Glyma18g02570), SEQ ID NO: 5 (Glyma18g02580), SEQ ID NO: 6 (Glyma18g02590), or SEQ ID NO: 7 (Forrest SNAP A111D mutant) and having Glyma18g02570, Glyma18g02580, Glyma18g02590, or SNAP activity, respectively.
 13. The transgenic progeny, seed, or plant part from the transgenic soybean of claim 1, wherein the transgenic progeny, seed, or part comprises the transcribable nucleic acid molecule.
 14. A soybean plant comprising in its genome: a) at least one introgressed allele locus associated with an SCN resistant phenotype wherein the locus is in a genomic region flanked by at least two loci selected from TABLE 6; and b) one or more polymorphic loci comprising alleles or combinations of alleles that are not found in an SCN resistant variety and that are linked to said locus associated with an SCN resistant phenotype, or a progeny plant therefrom.
 15. The soybean plant of claim 14, wherein the at least one allele locus is selected from the group consisting of Glyma18g02570, Glyma18g02580, and Glyma18g02590.
 16. The soybean plant of claim 14, produced by a method comprising: a) crossing a first soybean plant lacking a locus associated with an SCN resistant phenotype with a second soybean plant comprising: (i) an allele of at least one polymorphic nucleic acid that is associated with an SCN resistant phenotype located in a genomic region flanked by at least two loci selected from TABLE 6; and (ii) at least one additional polymorphic locus located outside of said region that is not present in the first soybean plant, to obtain a population of soybean plants segregating for the polymorphic locus that is associated with an SCN resistant phenotype and said additional polymorphic locus; b) detecting said polymorphic locus in at least one soybean plant from said population of soybean plants, and c) selecting a soybean plant comprising the locus associated with an SCN resistant phenotype that lacks the additional polymorphic locus, thereby obtaining a soybean plant comprising in its genome at least one introgressed allele of a polymorphic nucleic acid associated with an SCN resistant phenotype.
 17. The soybean plant of claim 16, wherein the first soybean plant comprises germplasm capable of conferring agronomically elite characteristics to a progeny plant of the first soybean plant and the second soybean plant.
 18. An artificial DNA construct comprising, as operably associated components in the 5′ to 3′ direction of transcription: (a) a promoter that functions in a soybean; (b) a transcribable nucleic acid molecule comprising (i) a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 (Glyma18g02570), SEQ ID NO: 2 (Glyma18g02580), and SEQ ID NO: 3 (Glyma18g02590), or a nucleotide sequence at least 95% identical thereto encoding a polypeptide having Glyma18g02570, Glyma18g02580, or Glyma18g02590 activity, respectively; (ii) a nucleotide sequence encoding a polypeptide comprising SEQ ID NO: 4 (Glyma18g02570), SEQ ID NO: 5 (Glyma18g02580), SEQ ID NO: 6 (Glyma18g02590), or SEQ ID NO: 7 (Forrest SNAP A111D mutant), or an amino acid sequence at least 95% identical thereto having Glyma18g02570, Glyma18g02580, Glyma18g02590, or SNAP activity, respectively; (iii) a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, wherein the polynucleotide encodes a polypeptide having Glyma18g02570, Glyma18g02580, or Glyma18g02590 activity, wherein said stringent conditions comprise incubation at 65° C. in a solution comprising 6×SSC (0.9 M sodium chloride and 0.09 M sodium citrate); or (iv) a nucleotide sequence which is the reverse complement of (i), (ii), or (iii); and (c) a transcriptional termination sequence.
 19. A method of increasing soybean cyst nematode (SCN) resistance of a soybean comprising: transforming a soybean plant with an artificial DNA construct according to claim 18; wherein the transformed soybean plant exhibits increased SCN resistance compared to a control not expressing the transcribable nucleic acid molecule. 